Department of 1 Medical Oncology, 2 Obstetrics and Gynaecology, and 3 Pathology and Laboratory Medicine, University Hospital, Groningen, The Netherlands
4 To whom correspondence should be addressed at: Department of Medical Oncology, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. e-mail: e.g.e.de.vries{at}int.azg.nl
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Abstract |
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Key words: cancer/fertility/ovary/tumour cell lysis
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Introduction |
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Purging, or clearing of minor quantities of tumour cells has been described in the haematopoietic stem cell transplantation setting (Champlin, 1996; Kvalheim et al., 1996
), but not for solid (tumour) tissue. Instead of ovarian cortex, a suspension of primordial or primary follicles may be autografted in plasma clots. This approach has restored estrogenic activity and fertility in a mouse model (Carroll et al., 1990
; Newton, 1998
). Possibly, making a suspension of follicles may allow purging of unwanted cell types in a similar way as for haematopoietic stem cells. A purging method, developed for haematopoietic stem cells by our institution, resulted in >99.9% tumour cell death without affecting colony formation by the stem cells (Schröder et al., 2000
). This method makes use of the bispecific antibody BIS-1, directed against the epithelial-related membrane antigen epithelial glycoprotein-2 [EGP-2, widely expressed on breast and ovarian carcinomas (De Leij et al., 1998
; Kroesen et al., 1998
)], and CD3 on T lymphocytes. BIS-1 creates functional cross-linking of T cells and tumour cells, allowing the delivery of a tumour cell-specific lethal hit inducing specific epithelial tumour cell death in vitro and in vivo (Kroesen et al., 1993
, 1994). This study was conducted to evaluate the concept of purging in the setting of a suspension of ovarian tissue.
Therefore, tumour cell death and morphological follicle survival were studied in an in vitro model in which activated lymphocytes and BIS-1 were added to the breast cancer cell line MCF-7, in the presence or absence of a suspension of human frozenthawed ovarian tissue. Two interactions were studied: (i) the effect of the purging process on the follicles; and (ii) the effect of the presence of an ovarian tissue suspension on the lymphocytetumour interaction, to determine whether this would reduce lymphocyte activity.
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Materials and methods |
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Thawing and suspension procedure. Ovarian tissue was thawed in a water bath at 37°C for 2 min maximum, and washed in a diminishing sequence of DMSO in Leibovitz medium with 10% fetal calf serum (FCS; Life Technologies). In order to obtain a cell suspension suitable for tumour cell purging, ovarian tissue was treated further in two steps. First, the tissue was mechanically dissected into a suspension with 27-gauge needles under sterile conditions. Then, enzymatic treatment was performed in medium containing 10 U/ml DNase I (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 300 U/ml collagenase IA (Sigma-Aldrich) for 2 h at 37°C. A combination of enzymatic treatment and microdissection has been used previously to suspend ovarian tissue (Oktay et al., 1997). The treatment appeared not to be toxic to tumour cells. The obtained cell suspension was transferred into RPMI medium (Life Technologies) with 10% FCS. Next, 100 µl of the cell suspension, was distributed into each well of a 96-well microtitre plate (Nunclon, Roskilde, Denmark). After overnight culture, the wells were inspected with an inverted phase-contrast microscope. In five independent experiments, the wells in which microscopically intact follicles were observed were counted. As the ovarian tissue suspension consisted of monocellular (stroma) cells and clumps of cells after enzymatic digestion, cell numbers were not standardized before purging. For wells in which follicles were present, the ovarian tissue suspension was carefully pipetted out and put into another 96-well plate in which chloromethyl fluorescein diacetate (CMFDA; Molecular Probes Europe BV, Leiden, The Netherlands)-labelled tumour cells were already present. These wells were used for purging experiments as described below.
Effector and target cells; bispecific antibody
Target cells: fluorescent detection system. The MCF-7 breast cancer cell line was used as a EGP-2-positive tumour model. Cells were plated in microtitre plates (Nunclon) and cultured overnight for optimal adhesion. Cells were labelled with the fluorescent dye CMFDA for 30 min. The optimal CMFDA concentration was established using concentrations from 0.5 to 15 µmol/l.
Cell detection was adequate at 1.5 µmol/l, without signs of cytotoxicity, and therefore this CMFDA concentration was used in subsequent experiments.
Effector cells. Lymphocytes were obtained from peripheral blood of healthy volunteers by Lymphoprep (Nycomed Pharma AS, Oslo, Norway) isolation. They were washed and incubated in vitro with anti-CD3 antibody (at 0.5 µg of IgG/ml RPMI medium with 10% FCS) for 72 h, and then washed and subsequently cultured in the presence of recombinant human interleukin-2 (rh IL-2; Aldesleukin, Chiron, Amsterdam, The Netherlands) at 100 IU/ml RPMI medium with 10% FCS for 48 h. Thereafter, cells were washed, counted and resuspended in RPMI medium with 10% FCS. This sequence of adding activating agents, as described above, was shown earlier to induce T lymphocyte activation (Schröder et al., 2000). The activated lymphocytes were used for purging experiments.
BIS-1. The BIS-1-producing quadroma was produced in our department by fusion of the hybridomas RIV-9 and MOC-31, producing anti-CD3 (IgG3) and anti-EGP-2 (IgG1) antibodies, respectively (De Lau et al., 1989). Preparation and purification were as described earlier (De Leij et al., 1998
). Briefly, BIS-1 was produced by means of a hollow fibre culture system (Endotronics, Minneapolis, MN). Purification of the hybrid antibodies (IgG3/IgG1) from parental-type antibodies, also produced by the quadroma, was performed by protein A column chromatography. BIS-1 F(ab')2 was then produced by means of digestion by pepsin followed by G150 Sephadex gel filtration, and added to a 0.9% sodium chloride solution to obtain a final concentration of 0.2 mg/ml.
Purging procedure
Tumour cell death, direct detection. Activated lymphocytes (effector cells) in the presence of 0.1 µg/ml BIS-1 were added to a 96-well plate (Nunclon), with 200, 500 or 1000 MCF-7 tumour cells (target cells) per well labelled with CMFDA, as described above. Ratios of effector to target cells were 100:1, 500:1 or 1000:1 (i.e. the addition of a 100-, 500- or 1000-fold more lymphocytes to the tumour cells), with an effector to target ratio of 0:1 (no lymphocytes added) as control. Co-incubation was performed in a total volume of 200 µl of RPMI medium with 10% FCS for 4 h at 37°C in a humidified, 5% CO2-containing atmosphere. The remaining tumour cells were counted directly by means of an inverted fluorescence microscope (Olympus IMT, Tokyo, Japan), at emission wavelength 516 nm, and excitation wavelength 492 nm. The results of the same effector to target ratios were taken together. Tumour cell death was assessed in five independent experiments.
The effect of the presence of the ovarian cortex suspension on tumour cell death efficiency was evaluated comparing tumour cell death as described above with tumour cell death in wells to which 100 µl of ovarian cortex suspension (prepared as described in Thawing and suspension procedure) was also added. Co-incubation was performed as described above. The prior fluorescent labelling of tumour cells allowed assessment of tumour cell death also in the presence of the ovarian cortex suspension. The effect of the addition of ovarian cortex suspension on tumour cell death efficiency was assessed with ovarian tissue of three patients.
The percentage tumour cell death was calculated as untreated tumour cells (control, effector to target ratio 0:1) minus the remaining tumour cells after treatment, divided by the control tumour cells, times 100%.
Tumour cell death, indirect detection. To evaluate longer term effects of the purging procedure on the growing potential of tumour cells, lymphocytes (effector cells) were co-incubated in a 96-well plate in the presence of 0.1 µg/ml BIS-1, with 2000 or 5000 MCF-7 tumour cells (target cells). Compared with the direct detection assay (see above), in this assay larger initial tumour cell numbers were necessary, in order to obtain an adequate formazan production in the MTT assay (see below) for measurement of tumour cell depletion (compared with the untreated control wells) after 5 days. The maximum total number of cells per well (due to culturing conditions in each well for 5 days) did not allow for higher effector to target ratios than used here, resulting in lower ratios than used in the direct detection assay.
Ratios of effector to target cells were 0:1, 10:1, 20:1 or 50:1, in a total volume of 200 µl of RPMI medium with 10% FCS (similar to that described in the experiment above). After 4 h of co-incubation at 37°C in a humidified, 5% CO2-containing atmosphere, the supernatant was removed. Cells were washed with fresh RPMI medium with 10% FCS and the supernatant was removed. Fresh medium (200 µl) was added and cells were cultured for 5 days (during which medium was refreshed one additional time) at 37°C in a humidified, 5% CO2-containing atmosphere. To establish tumour cell survival/growth after 5 days, the cellular reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) by the mitochondrial dehydrogenase of viable cells to a blue formazan product was evaluated in a standard microculture tetrazolium assay (Carmichael et al., 1987). DMSO (100%, 200 µl) was used to dissolve the formazan crystals, and the plate was read in an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Max, Molecular Devices, Sunnyvale, CA) at wavelength 490 nm. Comparisons were made with wells containing tumour cells and lymphocytes without BIS-1, tumour cells and BIS-1 without lymphocytes, or lymphocytes alone. In this experiment, no ovarian cortex suspension was added, as the described detection method does not allow discrimination between viable tumour cells or viable ovarian cells. Results of the same effector to target ratios were taken together. Depletion of growing tumour cells was assessed in four independent experiments.
The percentage depletion of growing cells was calculated as the number of untreated tumour cells (control) minus the number of remaining tumour cells after treatment, divided by the control number of tumour cells, times 100%. The background formazan production of lymphocytes was deducted.
Follicle morphology. Morphology and viability of follicles were assessed before freezing, after the freezingthawing procedure of tissue, and before and after the purging procedure. Before and after the freezingthawing procedure, pieces of tissue were fixed in buffered formalin, dehydrated through an alcohol series, and paraffin embedded. Before and after the purging procedure, ovarian cortex suspension was allowed to sediment onto glass slides, through a metal funnel-like device. Slides of all material were stained with standard Giemsa staining as well as the periodic acidSchiff method (PAS) (Whitaker and Williams, 1994), Papanicolau method (PAP) (Whitaker and Williams, 1994
) and the MOC31 antibody, directed against EGP-2, for tumour cell presence. Evaluation criteria for morphology on paraffin sections were eosinophilia of ooplasm, clumping of chromatin and wrinkling of the oocyte nuclear membrane as signs of atresia (Wright et al., 1999
).
Statistics
Tumour cell death, assessed by means of direct (fluorescent) detection or indirect (MTT) detection, was analysed by means of a two-sided Students t-test for independent samples. Also the effect of adding ovarian cortex suspension on tumour cell death was analysed by means of this test. All analyses were performed with the statistical software program SPSS. A P-value <0.05 was considered statistically significant.
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Results |
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Purging efficiency
Tumour cell death, direct detection. For each effector to target ratio, the proportion of tumour cells killed was the same, irrespective of the initial number of tumour cells (200, 500 or 1000). For instance, at an effector to target ratio of 1000:1 (i.e. 1000-fold more lymphocytes than tumour cells), tumour cell death for wells initially containing 200, 500 or 1000 tumour cells was 53 (n = 2), 46 ± 16 (n = 3) and 23 ± 7% (n = 3) without BIS-1 (P = NS); and 84 (n = 2), 79 ± 2 (n = 3) and 70 ± 14% (n = 3) with BIS-1 (P = NS). Tumour cell death was affected by BIS-1 (see below), but not significantly by the initial tumour cell count. Within each effector to target ratio (100:1, 500:1 and 1000:1, as well as the untreated control with effector to target ratio 0), we therefore pooled data for the three tumour cell concentrations (200, 500 or 1000 cells per well) before analysis. Figure 1A shows the percentage death of CMFDA-labelled tumour cells after lymphocyte-induced tumour cell death in the presence or absence of BIS-1 for 4 h. In the absence of BIS-1, increased tumour cell death is obtained with effector to target ratios of 500:1 and 1000:1 compared with the control with effector to target ratio 0, defined as 0% cell death. Adding BIS-1 to wells containing an effector to target ratio of 1000:1 further augmented this death (75.5% killed, SD 10.5, P = 0.002 compared with 40%, killed without BIS-1: SD 19). Tumour cell death is comparable with that in haematopoietic stem cell harvests (Schröder et al., 2000), with these effector to target ratios.
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Tumour cell death, indirect detection. In Figure 2, the percentage depletion of growing MCF-7 tumour cells is shown, after a 4 h co-incubation with activated lymphocytes in the presence or absence of BIS-1 and subsequent culture for 5 days. Depletion of growing tumour cells is clearly increased after treatment with activated lymphocytes in the presence of BIS-1, compared with the absence of BIS-1. In this setting, a maximum tumour cell depletion of 89% is seen at effector to target ratio of 10:1 (SD 11%, P < 0.001 compared with depletion without BIS-1).
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Effect of thawing and suspension procedure. The effect of the mechanical and enzymatic suspension procedure after thawing was evaluated in the ovarian cortex suspension after overnight culture, as described above. The mean number of wells (in the 96-well plate) microscopically containing one or more follicles was 62 ± 30 (SD; n = 5 replicates, see also thawing and suspension procedure). In these wells, no fibroblast outgrowth or attachment of granulosa cells was observed. The wells that were not used for purging experiments were used for cytology slides. Intact follicles were detected in these suspensions, and a representative sample of frozenthawed ovarian suspension is shown in Figure 3.
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Effect of purging. Morphological evaluation of the suspension including follicles, lymphocytes and tumour cells after the purging procedure, scored with the morphological criteria described above, revealed intact follicles remaining. A representative picture is shown in Figure 4. No MOC31-positive tumour cells were detected after the purging procedure.
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Discussion |
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The cryopreservation and thawing of ovarian tissue in this study was performed according to protocols described by the group of Gosden (Oktay et al., 1997). In the recent literature, seeding temperatures down to 9°C have been described, indicating that there is no optimal or standardized protocol for ovary cryopreservation so far (Gook et al., 2001
; Kim et al., 2001a
; Newton and Illingworth, 2001
; Radford et al., 2001
). The integrity of frozenthawed follicles after enzymatic isolation was confirmed by electron microscopy evaluation previously (Oktay et al., 1997
). Our results, showing morphologically intact follicles after thawing by light microscopy, are in line with this, although subcellular damage induced by freezingthawing cannot be excluded. We developed a fluorescent detection system to evaluate tumour cell depletion after the purging procedure. As our ultimate aim is to culture the suspension of ovarian tissue after the purging procedure, the Chromium51 release assay, commonly used to evaluate tumour cell depletion, was considered inadequate. With the fluorescent detection system, highly efficient tumour cell death by activated lymphocytes in the presence of BIS-1 was demonstrated. No (adverse) effect of the presence of ovarian tissue on tumour cell death was observed, and morphologically intact follicles were detected following the purging procedure. Therefore, this study supports the concept that solid tissue, rendered into suspension, can be purged in a similar way to haematopoietic stem cell material.
This study was performed to provide proof of principle, with morphological evaluation of the ovarian follicles. It is clear that future studies will have to address the important issue of the quantitative and functional survival of the follicles, according to studies performed by Hovatta et al. (Hovatta et al., 1997, 1999; Wright et al., 1999
). Ideally, in vivo evaluation of follicle functionality and tumour cell outgrowth would also follow, for instance by reinserting the follicles in a plasma clot under the renal capsule of SCID mice. Recently, the reinsertion of ovarian tissue resulted in growing and hormonally responsive primordial human follicles (Van den Broecke et al., 2001
). Furthermore, endogenous tumour cell infiltration of ovarian tissue instead of exogenously added tumour cells will have to be evaluated in this purging concept. As the enzymatic isolation of follicles most probably renders the endogenous tumour cells accessible for lymphocyte cell death, similar results to those with exogenous tumour cells are expected. It would be interesting to study whether (very) thin slices of ovarian tissue can also be purged in this way but, at present, no adequate in vitro model is available for the evaluation of the presence of residual tumour cells. To avoid potential non-specific lymphocyte activity directed against the ovarian tissue, the use of autologous patient lymphocytes will probably be preferred in a future patient-related setting, although no such activity was observed in this study. Previously, we already demonstrated that autologous patient serum does not affect purging efficiency (Schröder et al., 2000
), but for practical reasons (available volume) FCS was used in the present study. In reference to the cell model chosen in this study, one might argue that restoring endocrine function and fertility is undesirable in breast cancer patients, because of possible hormonal growth stimulation of residual disease. However, there is no evidence so far that a pregnancy after breast cancer treatment increases the risk of poor prognosis (Burstein and Winer, 2000
; Gelber et al., 2001
). With regards to the relevance of this study with a breast cancer cell model, it should be noted that a considerable number of breast cancer patients are diagnosed in their childbearing years. In The Netherlands, this amounts to ±1000 patients per annum;
10% of the women diagnosed yearly with breast cancer (Visser et al., 2000
). Together with the trend towards postponed childbearing (Heck et al., 1997
), preservation of fertility for these young cancer patients may become an issue of increasing importance.
In conclusion, this study provides a first step in the direction of purging cryopreserved ovarian tissue of tumour cells. This would imply that patients with an increased risk of tumour cell contamination of the ovary do not have to be excluded from gonadal cryopreservation beforehand. The safe replacement of ovarian tissue in female cancer survivors to restore their endocrine function and fertility would be a major step forward in the improvement of the quality of life for these women.
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Acknowledgements |
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Submitted on August 22, 2003; accepted on March 10, 2004.