The Use of Laser Microdissection and SELDI-TOF MS in Ovarian Cancer Tissue to Identify Protein Profiles

Materials and Methods

Ovarian cancer patients and tumor specimens. Tumor specimens were obtained from the historical tumor bank of the Department of Obstetrics and Gynecology, University Hospitals Leuven, Belgium. These were obtained after written informed consent and ethical approval from the local ethical committee and were handled according to protocol. Samples were obtained during primary surgery, snap frozen in liquid nitrogen after prelevation (delay between prelevation and freezing is less than 30 minutes) and stored at -80°C until further processing.

Medical records were reviewed for clinicopathological and follow-up data. Patients were identified as platinum resistant when the tumor recurred within 6 months after surgery followed by primary platinum based chemotherapy.

Determining optimal conditions for LMD of tissue samples and preparation of cell lysates. Cryostat sections (5 μm) were cut from frozen tissue samples on a Prosan cryostat at -20°C and mounted on a glass slide. These were subsequently stained with haematoxylin and eosin and were used as a control and orientation for tumor cell localization. Additional serial sections were made for LMD (16 μm thickness) and mounted on nuclease and human nucleic acid free membrane slides (polyethylene terepthlate (PET) membrane, 1.4 μm, MMI), which were stained with haematoxylin only and air dried. LMD was performed using the CellCut plus system (Olympus - MMI, Hamburg, Germany). Areas of necrosis, lymphocytic infiltration and regions with psammoma bodies were avoided.

To determine the number of cells needed to obtain a reliable profile with SELDI-TOF MS several numbers of cells varying from 5,000 to 80,000 were tested.

In order to extract a maximum amount of proteins several lysis conditions were investigated. Chemical lysis of these cells was performed comparing 3 different lysis buffers: (i) U9 buffer (urea 9M, CHAPS 2%, tris-HCl 50 mM, pH 9; Bio-Rad, Nazareth, Belgium) +/- complete protease inhibitor (Roche, Vilvoorde, Belgium); (ii) N-octyl glucoside 0.1%, urea 9 M, EDTA 1 mM (except for IMAC30 arrays), EGTA 1 mM, sodium orthovanadate 5 mM, sodium fluoride 10 mM and (iii) Complete lysis-M kit (Roche, Vilvoorde, Belgium). Temperature during lysis was tested ranging from lysis on ice, 4°C, room temperature to 70°C and duration of lysis ranged from 60 to 120 min. Subsequently several volumes of lysis buffer, ranging from 35 to 200 μL were tested.

LMD of 9 ovarian cancer tissue samples and preparation of cell lysates. For this study approximately 30,000 cells were dissected in quadruplicate, each in 30-60 minutes with adjusted settings for cutting speed, focus and laser energy to obtain a clear cut. Dissected cells were lysed and proteins extracted using 50 μL U9 buffer per 30.000 cells for 60 min at 4°C with shaking on a MicroMix 5 (DPC, UK). This mixture was then centrifuged for 5 min at 5,000 rpm and 4°C, diluted in the appropriate binding buffer according to the used ProteinChip (0.1M sodium phosphate, 0.5 M sodium chlorate for IMAC30; 0.1M sodium acetate pH 4.0 for CM10; 10% acetonitrile (ACN), 0.1% tri-fluoroacetic acid (TFA) for H50 and Tris-HCl 10-100 mM, pH 7.5-9 for Q10; Bio-Rad, Nazareth, Belgium) and filtered through a Nanosep MF device (0.2 μm; PALL inc, Haasrode, Belgium) to remove gross debris. This lysate was collected and stored at -80°C until MS analysis.

Protein profiling of laser microdissected cells with SELDI-TOF MS in 9 ovarian cancer patients. Protein lysates were analysed on copper-coated IMAC30 (immobilized metal affinity capture array), CM10 (weak cation exchanger), H50 (hydrophobic or reversed phase array) and Q10 (strong anion exchanger) arrays (Bio-Rad, Nazareth, Belgium). For the IMAC30 arrays, spots were pre-incubated twice with 50 μL of 0.1 M copper sulphate for 5 min at room temperature followed by a wash step with 0.1 M sodium acetate buffer pH 4 for 5 min at room temperature. For the H50 arrays spots were pre-washed twice with 50 μL of 50% ACN in ultrapure LC-MS grade water (Biosolve, Valkenswaard, The Netherlands) for 5 min at room temperature. Following these wash steps, and for CM10 and Q10 arrays immediately, spots were pre-incubated twice with array specific binding buffer followed by application of 100 μL of protein lysate and incubated for 60 min at 4°C with shaking on a MicroMix 5. After three additional wash steps with the same binding buffer and two final washes with water, 2×1 μL of 20% α-cyano-4-hydroxy cinnamic acid (CHCA, Bio-Rad, Nazareth, Belgium) dissolved in 1% TFA/100% ACN were applied.

Mass analysis was performed using SELDI-TOF MS (PCS 4000 Enterprise, Ciphergen ProteinChip Reader Inc., Fremont, CA, USA) according to an automated data collection protocol for a molecular weight range of 0-20,000 Da. The following settings were used: (a) laser intensity of 3,500 nJ; (b) focus mass 10,000 Da; (c) matrix attenuation 500 Da; (d) sampling rate 400 MHz; (e) 2 warming shots (not included in analysis), 10 data shots per point and (f) total number of points evaluated equal to 12.5% of the spot surface. Mass accuracy was calibrated externally using the all-in-one peptide standard according to the manufacturer's protocol (Bio-Rad, Nazareth, Belgium). A quality control sample (pooled serum) was analyzed weekly to validate the output of the system.

Using the Ciphergen Express Software, baseline subtraction and noise reduction were completed before peak intensities were normalized to the total ion current. Outlier spectra were identified and removed from analysis when the normalisation factor deviated more than 2 standard deviations. Numeric data were exported to Excel files for further biostatistical processing.

Background correction was performed for each sample separately and peaks were identified on the average of all samples (independent of platinum sensitivity) including all peaks with an absolute value ≥5.