PB from normal donors and patients diagnosed with AML, and leukapheresis products (from patients with lymphomas in remission without BM involvement) were collected after informed consent and in accordance with the University of Alberta Health Research Ethics Board guidelines. The clinical characteristics of 43 AML patients (26 male/17 female; mean age: 54 ± 16.8 years; mean% blasts in PB: 60.1%) are presented in Table 1. Light-density mononuclear cells (MNC) were separated using Ficoll-Paque PLUS (GE Healthcare, Baie d’Urfe, QC, Canada) and positively selected for CD34⁺ cells using immunomagnetic separation (Miltenyi Biotec, Auburn, CA, USA) as previously described [9]. The cells were washed with Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen, Burlington, ON, Canada), resuspended in serum-free media, and incubated at a concentration of 2 × 10⁶ cells/mL at 37 °C in 5% CO₂ for 48 h. For some experiments cells were treated with 20 ng/mL recombinant human (rh) TNF-α (Peprotech, Rocky Hill, NJ, USA). The cell-conditioned media were collected and evaluated by zymography or stored at −20 °C for ELISA. The cell pellets were used for RNA isolation or lysed and analyzed by Western blot. The human myelomonocytic leukemia cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD, USA) and grown in RPMI 1640 (Invitrogen) supplemented with 10% bovine growth serum (BGS, Hyclone, ThermoFisher Scientific, Nepean, ON, Canada). Colony forming unit-fibroblast (CFU-F) cultures were established from normal BM buffy coat cells as described [27]. Human umbilical vein endothelial cells (HUVEC) were cultured on gelatin-coated flasks in media consisting of M199, 10 mM L-glutamine, 250 IU/mL penicillin-streptomycin, 20% fetal calf serum (all from Invitrogen), and endothelial cell growth supplement (Collaborative Biomedical, Bedford, MA, USA). CFU-F and HUVEC were grown to sub-confluence, trypsinized, and plated in 24-well plates (1 × 10⁵ cells/mL). For co-culture experiments, PB MNC (2 × 10⁶ cells/mL) were seeded onto stromal monolayers, stimulated or not (control) with 20 ng/mL rh TNF-α and incubated for 48 h at 37 °C and 5% CO₂. Cell-conditioned media were evaluated by zymography.

Total RNA from AML PB MNC was extracted using TRIZOL (Invitrogen) according to the manufacturer’s instructions. RT-PCR reactions were carried out for MT1-MMP as described previously using GAPDH as internal control [28]. PCR products were electrophoresed through 2% agarose gels containing ethidium bromide and visualized under UV light using the FluorChem Imaging System; densitometric analysis was carried out using AlphaEase FCr image analysis software (Alpha Innotech, San Leandro, CA, USA). The relative level of target mRNA was regarded as the ratio between the intensities of the target primer and GAPDH bands. Real-time RT-PCR was performed as previously described [28]. Quantitative assessment of MT1-MMP mRNA levels was performed using an ABI PRISM^® 7000 Sequence Detection System (ABI, Foster City, CA, USA) and primer (Quantitect Hs_MMP14_1_SG) from Qiagen (Toronto, ON, Canada). A 25 μL reaction mixture containing 12.5 μL Quantifast SYBR Green PCR master mix (Qiagen) and 10 ng of cDNA template, forward and reverse primers, were used. The threshold cycle (Ct), i.e., the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was subsequently determined. Relative quantitation of MT1-MMP mRNA expression was calculated using the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control gene and relative to a calibrator, is expressed as 2-∆∆Ct (fold difference), where ∆Ct = (Ct of target gene [MT1-MMP]) – (Ct of endogenous control gene), and ∆∆Ct = (∆Ct of samples for target gene) – (∆Ct of calibrator for the target gene).

Cell pellets were sonicated in lysis buffer (1% Triton, 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulphonyl fluoride, 1 mM Na₃VO₄, 5 μg/mL leupeptin, 5 μg/mL aprotinin, and 0.5 μg/mL pepstatin A, pH 7.3; all reagents from Sigma, Oakville, ON, Canada) and the lysates clarified by centrifugation at 18,000 × g for 10 min at 4 °C. Protein concentration was determined using the Bio-Rad protein assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA), and equal amounts of protein (30 μg) were loaded in each well. Samples were resolved in a 10% polyacrylamide gel under reducing conditions and transferred to a nitrocellulose membrane. Following blockage with 5% fat-free dried milk in Tris-buffered saline and 0.05% Tween 20, the membrane was probed with rabbit anti-human MT1-MMP polyclonal antibody (Abcam, Cambridge, MA, USA) and a secondary antibody (goat anti-rabbit, HRP-conjugated IgG, Pierce Biotechnology, Rockford, IL, USA). Chemiluminescence detection was performed using the Supersignal West Pico system (Pierce). The intensity of the bands was analyzed by densitometry using the AlphaEase FCr image analysis software (Alpha Innotech).

Cell invasion was determined using the trans-Matrigel assay as we described previously [9,29]. Briefly, 13 mm polycarbonate filters (Costar/Nucleopore, Toronto, ON, Canada) were coated with 25 μg of Matrigel (Collaborative Biomedical Products) and placed between the upper and lower compartments of modified Boyden chambers (Neuro Probe Inc, Gaithersburg, MD, USA). The lower chambers contained IMDM supplemented with 0.1% BSA. AML PB MNC treated or not (control) with 20 ng/mL rh TNF-α were loaded onto the upper compartments (2 × 10⁵ cells/chamber) and incubated overnight at 37 °C, 5% CO₂. In some experiments MNC were pre-incubated (1 h) with various concentrations of (−)-epigallocatechin 3-gallate (EGCG, Sigma) or 1 μg/mL Enbrel (Amgen, Missisauga, ON, Canada) before rh TNF-α treatment (2 h). Cells that had migrated through the Matrigel-coated filters were recovered from the lower compartments and counted using a Neubauer hemocytometer. Percentage migration was calculated from the ratio of cells recovered from the bottom chambers to the total number of cells loaded. The assay was performed at least in quadruplicate for each condition and each sample tested.

Small interfering (si)RNA was targeted against 21-nucleotide sequences of MT1-MMP (Dharmacon, Lafayette, CO, USA) as described [18,30]. A control siRNA sequence was generated from a scrambled MT1-MMP sequence. THP-1 cells (5 × 10⁶) were electroporated with siRNA oligonucleotides (5 μg) using a nucleofector kit (Amaxa, Gaithersburg, MD, USA) according to the manufacturer’s instructions and incubated for 24 h in RPMI + 10% BGS at 37 °C and 5% CO₂. Viability of the transfected cells as determined by the trypan blue exclusion test was >80%. The efficiency of MT1-MMP knockdown was determined by flow cytometry.

Cells were labelled with rabbit anti-human MT1-MMP antibody (Millipore) for 45 min at 4 °C, and then stained with goat anti-rabbit IgG-555 (Invitrogen). The cells were then fixed in 1% paraformaldehyde and analyzed by flow cytometry (FACS Calibur, Becton Dickinson, San José, CA, USA) and FCS Express V3 (De Novo Software, Los Angeles, CA, USA).

The gelatinolytic activities in cell-conditioned media were assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as we described [9]. Briefly, conditioned media were applied onto a 12% SDS-PAGE gel containing 1.5 mg/mL gelatin (Sigma) alongside media conditioned by fibrosarcoma HT-1080 cells, which exhibit both the active and pro- forms of MMP-2 and MMP-9. Clear bands at 92 kDa, 72 kDa and 62 kDa against a Coomassie blue background indicated the presence of proMMP-9, proMMP-2 and active MMP-2, respectively.

TNF-α levels were measured by ELISA (Peprotech) in media conditioned by PB MNC from normal donors and AML patients as described above. Briefly, a 96-well plate was coated with 1 μg/mL anti-human TNF-α antibody and incubated overnight at room temperature. The next day, the wells were washed (0.05% Tween-20 in PBS) and block buffer (1% BSA in PBS) added. Standards and samples (100 μL) were loaded onto wells in triplicate, the plate was incubated overnight at room temperature, and after washing, detection antibody (0.5 μg/mL) was added. For color development, avidin peroxidase conjugate and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS, both from Sigma) were used, and optical was density measured at 405 nm with background correction at 650 nm.

Arithmetic means and standard deviations were calculated and statistical analysis of significance was carried out using a paired, 2-tailed Student’s t-test with 95% confidence interval. Calculated p-values between data sets were considered significantly different when p ≤ 0.05.

Transcripts for MT1-MMP were found in 95% (41 out of 43) of AML samples tested (Table 1 and representative data in Figure 1A). MT1-MMP protein, as evaluated by Western blotting, was strongly expressed in AML cells (Figure 1B) in contrast to the weaker expression shown previously by us in normal BM MNC and CD34⁺ HSPC [18]. Here we also found, using quantitative RT-PCR analysis, that both steady-state and mobilized MNC and mobilized CD34⁺ cells from normal PB have significantly lower MT1-MMP mRNA expression compared to MNC from AML PB (Figure 1C).

Previously, we reported that AML blasts (from 15 patients diagnosed with AML) express MMP-9 and MMP-2 at the gene and protein levels [22]. Here, we extended our study to 43 additional patients diagnosed with AML (Table 1). We confirmed that AML cells strongly secrete proMMP-9 and various levels of proMMP-2, while monocultures of stromal cells (BM fibroblasts or endothelial cells) secrete only proMMP-2 [22]. Furthermore, we show here that when AML MNC were co-cultured with stromal cells, not only are the levels of proMMP-2 increased, but the active form (MMP-2) appeared (Figure 1D). Whereas active MMP-2 was undetectable in serum-free media conditioned by AML MNC alone, it was observed in co-cultures of stromal cells with MNC from two patients (#16 and #18) that weakly secrete proMMP-2, and from one (#24) that did not. Moreover, we recently reported that co-cultures of fibroblasts with normal BM CD34⁺ cells, which do not secrete proMMP-2, show active MMP-2 [18]. This suggests that proMMP-2 secreted by stromal cells can be activated by MT1-MMP secreted by hematopoietic cells and both are necessary for the proteolytic cascade leading to proMMP-2 activation [10,22].

We silenced the gene expression of MT1-MMP in leukemic THP-1 cells with siRNA. The expression of MT1-MMP in transfected cells was 50% of that in cells transfected with scrambled (control) siRNA (Figure 2A). We performed co-culture experiments using THP-1 cells that were pre-treated with MT1-MMP siRNA and found (using zymography) significantly reduced activation of proMMP-2 in conditioned media from co-cultures of these cells with fibroblasts in comparison to co-cultures with control scrambled siRNA THP-1 cells (Figure 2B). When THP-1 cells were evaluated in the trans-Matrigel migration assay, a significant reduction in migration of the MT1-MMP siRNA-transfected THP-1 cells was observed compared to control (scrambled siRNA) cells (Figure 2C).

Moreover, when we exposed conditioned media from co-cultures of AML cells and fibroblasts to EGCG, an inhibitor of MT1-MMP and other MMPs [31], we observed a decrease in the level of proMMP-9 and proMMP-2 as well as a dose-dependent (from 1 to 50 μM of EGCG) reduction of proMMP-2 activation (Figure 2D). EGCG had no significant effect on cell viability (data not shown). We also found that EGCG reduces the migration of AML MNC across the Matrigel in a dose-dependent manner (Figure 2E), and percentage migration was decreased by 50% to 90% using 50 μM EGCG (Figure 2F).

Previously, we reported that rh TNF-α strongly upregulates the secretion of proMMP-2 and proMMP-9 in normal human HSPC [9]. Here using ELISA, we evaluated the level of TNF-α in media conditioned by AML MNC. We found significantly higher mean levels of TNF-α in media conditioned by AML (n = 12) compared to normal MNC (n = 3) (Figure 3).

Having confirmed an earlier report that TNF-α is elevated in AML [26], we next examined whether TNF-α has any effect on MT1-MMP mRNA expression. We found that rh TNF-α increased (up to 1.4-fold by gel-based RT-PCR and up to more than 3-fold by quantitative RT-PCR) the expression of MT1-MMP transcripts in MNC from AML patients (Figure 4A,B, respectively). The expression of total MT1-MMP protein, as evaluated by Western blotting, was increased up to 1.4-fold (Figure 4C). The moderate increases in MT1-MMP gene and protein expression upon TNF-α stimulation could be explained by the fact that these cells had been pre-exposed to endogenously produced TNF-α. When we stimulated AML cells with rh TNF-α under co-culture conditions with fibroblasts, we observed a very strong activation of proMMP-2, although TNF-α barely affected the secretion of proMMP-2 by BM stromal fibroblasts or AML blasts in monocultures (Figure 4D). In addition, we observed that TNF-α also increased proMMP-9 secretion (5.5-fold) when AML MNC were co-cultured with fibroblasts.

Moreover, rh TNF-α stimulated the trans-Matrigel migration of AML MNC. This trans-Matrigel migration was inhibited by EGCG, as well as by Enbrel, a soluble TNF-receptor fusion protein that blocks TNF-α. Together EGCG and Enbrel had additive inhibitory effects on the trans-Matrigel migration of AML MNC stimulated with TNF-α (Figure 4E).