Plasma levels of the MMP-9:TIMP-1 complex as prognostic biomarker in breast cancer: a retrospective study

Plasma concentrations of MMP-9:TIMP-1 complex were assayed using a commercially available ELISA (DuoSet® ELISA Development System, R&D Systems, R&D Systems Europe Ltd., United Kingdom; Human MMP-9:TIMP-1 complex catalog number DY1449) according to the manufacturer’s instructions. Before assaying plasma samples, the assay was validated for measurement of complexes in plasma. The validation process included investigations of reproducibility (intra-and inter-assay), recovery, and linearity upon dilution of samples. Recovery was determined by adding a fixed amount (1 ng/mL) of recombinant MMP-9:TIMP-1 complex to a plasma dilution series. Subsequently, recovery was calculated as the measured amount of complex in relation to the expected amount in each sample and reported in per cent. Intra-assay variation was determined by measurements of identical samples of a plasma pool diluted 3 times in reagent diluent buffer on the same plate. Inter-assay variation was determined by including duplicates of dilutions of the plasma pool on every plate measured. Linearity of signal was determined by measuring the signal in a dilution series of plasma (two-fold serial dilution ranging from 1-4000 times). Finally, the limit of detection was determined by repeated measurements of a blank sample and calculated as the mean of the signal in these blank samples plus three standard deviations. For validation purposes, a plasma pool obtained from a healthy donor plus a pool of plasma from cancer patients were used. For analysis, the plasma samples were diluted 3 times.

The proximity probes directed against MMP-9 and TIMP-1 were prepared by linking a single batch of affinity purified polyclonal antibody (MMP-9 (R&D Systems, Cat.no. AF911) or TIMP-1 (R&D Systems, Cat.no. AF970)) to 3’-hydroxyl free and 5’-phosphate free 40-mer oligonucleotide sequences (Figure 1). Thereby, the unique amplicons are forming, which are representative for each target protein. The antibody-oligonucleotide conjugates were generated by Innova Biosciences (United Kingdom) using the Lightning-Link^TM technology. Conjugation quality was analyzed by SDS-PAGE.

The basic PLA protocol has previously been described in details [30]. In brief, 2 μL of a plasma sample was mixed with 2 μL of plasma dilution buffer (Olink Bioscience, Sweden) including 200 pM of GFP as internal control standard spike-in. The mix was incubated at room temperature for 20 minutes. Probe mixture containing Probe Mix Diluent (Olink Bioscience), 1% BSA (Calbiochem/Merck, Germany) and 0.1% Triton X-100 (Merck, Germany) was added to each sample in a 1:1 ratio and incubated at 4°C overnight. Then 4 μL probed sample was mixed with 96 μL Ligation reaction buffer (Olink Bioscience, available upon request) containing 100 nM connector oligonucleotide and 0.0006 units of T4 DNA ligase (Fermentas, USA). The ligation was achieved by incubation at 37°C for 10 minutes, followed by 10 minutes of heat inactivation at 65°C. Prior to pre-amplification the connector oligonucleotide was digested using 1 unit of uracil-DNA excision mix (Epicenter, USA). Pre-amplification was performed in a total volume of 25 μl by mixing 20 μl of the ligated product with 5 μL PCR mix (1× PCR buffer (Invitrogen, Denmark), 15 mM MgCl₂ (Invitrogen), 1 mM dNTP (Invitrogen), 0.2 μM of each forward and reverse pre-amplification primer (Figure 1) (Biomers, Germany), and 7.5 units Platinum Taq polymerase (Invitrogen)), using the same amplification protocol as previously described [30]. Prior to qPCR, the products were diluted 5-fold in 1× Tris-EDTA buffer.

qPCR was performed on a LightCycler 480 (Roche, Denmark) using a 384-well format. The diluted DNA products were mixed with 1.4× Fast Universal Master Mix (Life Technologies, Denmark), dH₂O and 0.05 units of uracil-DNA excision mix (Epicenter) and incubated for 30 minutes at 37°C to digest any leftover primers in the solution. Seven μL of each sample was then transferred to each well and mixed with 3 μL of 3 μM primer (Figure 1) (Biomers), dH₂O and 0.8 μM TaqMan probe (Life Technologies, Denmark) to a total sample volume of 10 μL per well. The thermal cycler program was initiated with 5 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. The time period from initiation of PLA measurements until end of all incubations was four weeks.

We initiated our validation of the PLA technique as a tool for measuring protein:protein complex in biological samples with some basic experiments of protocol optimization. The performances of the MMP-9 and TIMP-1 probes were assessed by their ability to measure increasing concentration of the recombinant proteins. MMP-9 (R&D Systems, Cat.no. 911-MP-010), TIMP-1 (in-house, purified as [31]), and complexed MMP-9 and TIMP-1 in PBS + 0.1% BSA were tested. Probe concentrations of 50, 75, 100 and 200 pM were tested in order to establish the optimal assay setup, which gave the lowest background signal and best linear range for each of the two possible probe combinations. The incubation time and temperature were tested in two different settings, one hour at 37°C and overnight at 4°C. The effect of pre-amplification was tested in both buffer and plasma to explore if a less time-consuming protocol could be applied. Unspecific annealing of the qPCR primers was tested by incubation with the MMP-9:TIMP-1 probes and recombinant protein, followed by qPCR on the resulting amplicon with primers for the total MMP-9, total TIMP-1 or specific primers for the MMP-9:TIMP-1 complex as positive controls. The dynamic range of the MMP-9:TIMP-1 complex measurements in human plasma was evaluated by selecting four different plasma samples from breast cancer patients. Using the ELISA measurements we selected two samples with a low level of TIMP-1 and two with a high level of TIMP-1. This was done to investigate if the decrease in signal was correlated with the dilution factor, but also to find the optimal dilution range of the plasma samples. The precision of the assay was investigated by analysis of these four samples, four times, on four different days. Every day, the samples were handled independently and the standard curves were freshly made. The measurements were made in quadruples, separated prior to the qPCR. This gave an estimate of the intra-assay variation within a run on the qPCR plate. The measurement between days allowed for an estimate of the inter-assay variation of the assay. For analysis, the plasma samples were diluted 50 times.

For internal control standard we used recombinant green fluorescent protein (GFP) (Vector Laboratories, USA), which was spiked in the plasma dilution mix (Olink Bioscience) and thereby added to all sample incubations. GFP was diluted in PBS + 0.1% BSA (Calbiochem/Merck, Denmark) and mixed with the plasma diluent mix to a final concentration of 10 nM. We used the data from GFP to evaluate the technical performance of the PLA runs. A standard curve of the MMP-9:TIMP-1 complex was made to investigate the linear range of the assay. Also, the standard curve was used for calculation of the MMP-9:TIMP-1 concentration of each sample. The recombinant MMP-9 and TIMP-1 were mixed in PBS + 0.1% BSA to create the complex standard curve; this ranged from 0.010 to 10 nM. The standard curve was prepared and went through a PCR run and then divided into aliquots and stored at -20°C until qPCR run; these standard curves were loaded to each qPCR plate. Recovery and specificity studies were made in both PBS + 0.1% BSA, chicken plasma (GeneTex, USA, Cat.no. GTX73211) and human plasma. Recombinant proteins were spiked in these three different materials in the range of 200 pM to 2000 pM.