Patients
Mitochondrial density was analysed in 24 borderline tumours, 155 primary ovarian cancers, and 26 recurrent ovarian cancer samples. Formalin-fixed paraffin-embedded (FFPE) samples were obtained from the files of the University Medical Center Hamburg-Eppendorf. These were collected during surgeries from patients with primary and recurrent disease treated between 2016 and 2020. The FFPE samples from the Würzburg University Hospital were collected between 1990 and 1997. The cohorts contain tissue from different locations such as the fallopian tube, the ovary itself and metastatic sites (omentum and peritoneal wall). The histological and clinical data of both collectives are shown in Table
1.
Table 1
Descriptive statistics of the cohort
Borderline tumour | | 17 (12%) | | 7 (11%) |
Recurrent ovarian cancer | | 20 (14%) | | 6 (9%) |
Primary ovarian cancer | | 102a (74%) | | 53 (80%) |
Age at diagnosis (y) | Mean (median) | 59.3 y (60 y) | | 56.5 y (58.5 y) |
Histological type | Serous papillary | 93 (67%) | | 62 (94%) |
Endometrioid | 2 (1%) | | 1 (2%) |
Clear cell | 1 (0,7%) | | 1 (2%) |
Müllerian mucinous | 5 (4%) | | 2 (3%) |
FIGO state | FIGO I-II | 2 (1%) | | 30 (45%) |
FIGO IIIA-IIIB | 6 (4%) | | 10 (15%) |
FIGO IIIC | 69 (50%) | | 22 (33%) |
FIGO IV | 17 (12%) | | 4 (6%) |
Nodal involvement | Negative | 14 (10%) | | |
Positive | 54 (39%) | | |
Grading | Low grading | 3 (2%) | | 19 (29%) |
High grading | 85 (61%) | | 47 (71%) |
Distant metastasis | Negative | 72 (52%) | | 62 (94%) |
Positive | 20 (14%) | | 4 (6%) |
Tumour residuum after surgery | Not macroscopically visible | 53 (38%) | | 33 (50%) |
< 1cm3 | 23 (17%) | | 5 (8%) |
> 1cm3 | 25 (18%) | | 28 (42%) |
Adjuvant chemotherapy | Carboplatin/Paclitaxel (Taxol) | 97 (70%) | Cyclophosphamide/Carboplatin | 10 (15%) |
Bevacizumab | 61 (43%) | Carboplatin mono | 3 (5%) |
| | Cyclophosphamide/Cisplatin | 36 (55%) |
| | None | 17 (26%) |
Recurrence | Yes | 70 (50%) | | 14 (21%) |
No | 32 (23%) | | 35 (53%) |
| | Inoperable | 17 (26%) |
Recurrence-free survival (month) | Mean (median) | 23.88 (21) | | 30.07 (22) |
Overall survival (month) | Mean (median) | 36 (33.5) | | 39.09 (19) |
All patients of the Hamburg cohort provided written informed consent prior to their biological material and clinical records being accessed according to the guidelines of the Review and Ethics Committee (#190504 and PV6012) of the University Medical Center Hamburg-Eppendorf [
19]. The histological sections have been used in a previous study where the regulatory approvals are described [
20]. Their clinical data were obtained from a detailed institutional database containing information on surgical, therapeutic and clinicopathological procedures [
19]. The clinical data of the Würzburg cohort as well as the associated biosamples were used in anonymised form. This complies with ethical and current legal requirements requested by the Ethics Committee of the University of Würzburg. This study is also fully compliant with the Declaration of Helsinki.
Cell lines and cell culture
Human ovarian cancer cell lines OVCAR8 (Medium uses for OVCAR8: Roswell Park Memorial Institute (RPMI-1640) medium (Gibco)), SKOV3 (Medium used for SKOV3: McCoy’s 5a Medium Modified (Gibco)), OVCAR3 (Medium uses for OVCAR3: Roswell Park Memorial Institute (RPMI-1640) medium (Gibco)) and COV644 (Medium used for COV644: DMEM (Gibco)) were analysed. All four cell lines grew as adherent cells. The medium was supplemented with 10% (SKOV3, OVCAR8, COV644) or 20% (OVCAR3) fetal calf serum (FCS) and 2 mM L-glutamine, 100 units/mL penicillin, 100 g/mL streptomycin (GibcoTM, Waltham, MA, USA) as seen in Table
2. Cells were maintained in a humidified atmosphere of 95% air and 5% CO
2 at 37 °C. If confluence reached 70–80%, cells were passaged to expand them. Subsequently, immunohistochemical detection was performed and evaluated from in vitro cultured SKOV3, OVCAR3, and COV644 ovarian cancer cells fixed in formalin and embedded in wax (FFPE) as described below.
SKOV3 | Serous cystadenocarcinoma | Ascites | ATCC® HTB77™ | McCoy’s 5A + 10% FCS and 1% P/S, 2 mM L-Glu |
OVCAR3 | Poorly differentiated papillary Adenocarcinoma | Ascites | D. Thormeyer, Fachklinik Hornheide | RPMI + 20% FCS and 1% P/S, 2 mM L-Glu |
OVCAR8 | Adenocarcinoma | Ovary | V. Aßmann, UKE Hamburg | RPMI + 10% FCS and 1% P/S, 2 mM L-Glu |
COV644 | Mucinous ovarian carcinoma | Solid tumour, ovary | ECACC 07071908 | DMEM + 10% FCS and 1% P/S, 2 mM L-Glu |
Immunohistochemistry (IHC)
Four µm thick FFPE sections were deparaffinised in two changes of xylene (5 min each), rehydrated in a series of graded ethanol (100%, 96%, 70% and 50% for 5 min each) and finally washed for two min in distilled water. For antigen retrieval, sections were pre-treated with a steamer (Dako Cytomation, Carpinteria, CA, USA) for 20 min at 100 °C in the case of anti-heat shock protein (HSP) 60 (NBP1-77,397, 1 mg/mL, Novus Biologicals, Colorado, USA), 99 °C for 10 min for anti-fumarase (NBP1-31,336, 0,9 mg/mL, Novus Biologicals, Colorado, USA) or 125 °C for 4 min in the case of anti-succinic dehydrogenase antibody (SDH; HPA041981, Atlas Antibodies, Bromma, Sweden) in a 1:10 dilution of epitope retrieval solution (#S1699, pH 6,0 Dako, Carpinteria, CA, USA). After cooling, the sections were rinsed twice with Tris-buffered saline/0.1% Tween20 (TBS-T) and once with TBS (pH 7.6) for 5 min each. The performance of the following steps took place in a humid chamber. The incubation was done with either anti-HSP 60 antibody (#NBP1-77397, Novus Biologicals), diluted 1:400 in Antibody Diluent (#S0809, Dako Carpinteria, CA, USA), or anti-fumarase antibody (#NBP1-31336, Novus Biologicals) diluted 1:1000 in Antibody Diluent or anti-SDH antibody (#HPA041981, Atlas) diluted 1:50 in Antibody Diluent at room temperature (RT) for 60 min. Rabbit poly IgG antibody (#ab37415, abcam, Berlin, Germany) diluted 1:2000 (HSP60), 1:5556 (fumarase) or 1:834 (SDH) in Antibody Diluent was used as an isotype control. After incubation, the sections were rinsed twice with TBS-T and once with TBS for 5 min each. Next, the sections were incubated with a secondary biotin-conjugated swine-α rabbit antibody (#E0353, Dako, Carpinteria, CA, USA) diluted 1:200 in TBS for 30 min at RT. This was followed by another washing step with TBS and TBS-T for 5 min each.
Subsequently, the sections were incubated with Vectastain® ABC-AP kit (#AK5000, Linaris, Dossenheim, Germany) according to the manufacturer’s recommendation at RT for 30 min followed by washing again in TBS-T and TBS as described above. The sections were incubated with the Permanent Red solution (#ZUC001-125, Zytomed Systems GmbH, Berlin, Germany) for 10 min (HSP60), for 20 min (Fumarase) and for 12 min (SDHA), respectively, to visualise the alkaline phosphatase enzyme activity. Subsequently, counterstaining was performed with hemalumn for 8 s and the sections were rinsed under running tap water for 5 min and in Aqua dest for 2 min.
FFPE embedded human kidney served as a positive control as mitochondria are abundantly present in the proximal convoluted tubule of the kidney.
Finally, the slides were dehydrated in a series of graded ethanol (70% for 15 s, 96% and 100% for 5 min each) and three changes of xylene (5 min each) and coverslipped with Eukitt® mounting medium (#03989, Sigma-Aldrich, Taufkirchen, Germany).
Microscopy and image analysis
The sections were visually analysed using a ZEISS Axiophot 2 microscope (Carl Zeiss, Jena, Germany). After the initial visual control, the sections were digitised using a ZEISS AXIO Scan Z1 slide scanner equipped with a ZEISS EC Plan-Neofluar 20 × /0.50 pole M27 objective (Carl Zeiss, Jena, Germany) and a Hitachi HV-F20SCL 1600 × 1200 pixels camera (Hitachi Kokusai Electric America Ltd., New York, NY, USA). ZEISS ZEN 2.3 software (Carl Zeiss, Jena, Germany) was used for image acquisition. The netScope Viewer software (Net-Base Software, Freiburg, Germany) was used for further image processing.
The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low, intermediate, and high.
Flow cytometry with MitoTracker Green
All four cell lines (see Table
2; at ~ 80% confluency) were trypsinised and afterwards centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the remaining pellets were re-suspended in 100 µL of pre-warmed (37℃) 60 nM MitoTracker™ Green FM (Thermo Fisher Scientific, USA) prepared according to the manufacturer’s instructions.
The cells were then incubated for 30 min under standard cell culture conditions (at 37℃ in 5% CO2, 95% humidity). The incubation was followed by a washing step, using flow cytometry buffer (1% BSA, 0.05% NaN3 in Dulbecco's phosphate-buffered saline). After centrifugation, the cell pellets were re-suspended in flow cytometry buffer, and the fluorescence was measured using the BD FACSCanto II Flow Cytometer. Propidium iodide (PI) (1 µg/mL) was used to stain the dead cells. The analysis of the flow cytometry was done using FlowLogic software (Inivai, Mentone Victoria, Australia).
Statistical analyses
Statistical analyses were performed using Microsoft Excel version 16.47 (One Microsoft Way, Redmond, WA, USA). IHC sections were semi-quantitatively classified into low ( +), middle (+ +) or high (+ + +) mitochondrial content. The possible correlation between this quantification and histology (borderline, primary ovarian cancer and recurrent ovarian cancer) and clinic-pathological factors (Fédération Internationale de Gynécologie et d'Obstétrique (FIGO)-Classification, tumour stage, nodal involvement, histological subtype, grading and postoperative residual tumour) was analysed. Survival curves were analysed using GraphPad PRISM 9 (GraphPad Software, San Diego, USA). In addition, we conducted a multivariate analysis to include other clinical parameters. This was done with SPSS software Version 25 (IBM SPSS Statistics, Armonk, NY, USA). The correlations between mitochondrial content, survival and clinicopathologic factors (FIGO and residual postoperative tumor) were analysed using a multivariate Cox proportional hazards regression model. Probability values (p values) less than 0.05 were considered statistically significant.