Matrix metalloproteinase-2 (MMP-2) is important in the dissemination and invasion of tumor cells and activates angiogenesis. We present an immunocytochemical study of MMP-2 expression in circulating prostate cells (CPCs), disseminated tumor cells (DTCs), and micrometastasis (mM) in bone marrow of men with prostate cancer.
With the increasing use of prostate specific antigen as a screening test to detect prostate cancer, the frequency of men presenting with metastatic disease has decreased [
Cancer cells disseminate from the original cancer, first to the neurovascular structures and then to the blood [
If, as the reports indicate, increased MMP-2 expression in the primary tumor is associated with a worse prognosis, one explication could be that cells expressing MMP-2 disseminate early to distant tissues, are not therefore affected by loco-regional treatments, and as a consequence are able to develop into metastasis. If this hypothesis is correct, circulating tumor cells should express MMP-2 whether they are circulating in blood or the bone marrow, and MMP-2 expression would permit the invasion of the endostium and therefore facilitate the formation of micrometastasis. The coexpression of PSA and MMP-2 in bone marrow fragments would confirm this hypothesis.
These data prompted us to investigate the expression of MMP-2 in circulating prostate cells in blood and bone marrow, and in the micrometastasis in bone marrow fragments in a population of patients with prostate cancer, after radical prostatectomy, both in patients bone scan negative and positive.
The transverse population included patients diagnosed with prostate cancer attending the Hospital de Carabineros de Chile and the Instituto of BioOncología, Santiago, Chile between 2008 and 2011. Patient records were used to retrieve clinical information (age, stage, Gleason score, treatment, bone scan results, and serum PSA at the time of sampling).
The following is definition of circulating prostate cells (CPCs), disseminated tumor cells (DTCs) in bone marrow aspirates, and micrometastasis (mM). The criteria of ISHAGE were used to evaluate immunostained cells [ CPCs: secondary CPC: detected in blood after radical treatment (Figure DTCs: cells detected in bone marrow aspirates or bone marrow biopsy touch preparations, but not in bone marrow fragments (Figure Micrometastasis: cells detected in bone marrow fragments from biopsy specimens (Figures
(a) PSA (+) CPC. (b) circulating leucocytes.
Disseminated tumor cell.
(a) Micrometastasis PSA (+). (b) Biopsy PSA (−). (c) Borders of microfragment MMP-2 (+). (d) Central pattern MMP-2 (+) stromal cells MMP-2 (+). (e) Central pattern MMP-2. (f) Microfragment MMP-2 (−), surrounding stromal cells MMP-2 (−). (g) Borders of microfragment MMP-2 (+), surrounding stromal cells MMP-2 (−). (h) Microfragment MMP-2 (−), stromal cells MMP-2 (−), DTC MMP-2 (+). (i) Borders of microfragment MMP-2 (+), some stromal cells MMP-2 (+).
Biopsy proven prostate cancer. Written informed consent. Bone scan within three months of the sampling.
After written informed consent bone marrow samples were obtained by an aspiration (4 mL) and a biopsy from the posterior superior iliac crest, and an 8 mL venous blood sample was taken at the same time.
4 mL aspirate sample of bone marrow and 8 mL of blood were collected into EDTA (Beckinson-Vacutainer) and processed within 30 minutes. The sample was layered onto 2 mL Histopaque 1.077 (Sigma-Aldrich) at room temperature. The mononuclear cells were obtained according to manufacturer’s instructions and finally washed 3 times in phosphate buffered saline pH 7.4 (PBS). The pellet was resuspended in 100
The bone marrow biopsy sample was used to make 3 “touch-preps” using silanized slides (DAKO, USA) and fixed as previously described.
Monoclonal antibodies directed against PSA clone 28A4 (Novacastro, UK) in a concentration of 2,5
Positive samples underwent a second stage of processing, using the monoclonal antibody against MMP-2 clone 1B4 (Novocastra, UK) and a system of detection based on peroxidase (LSAB2, DAKO, USA) with DAB (DAKO, USA) as the chromogen. Endogenous peroxidase was inhibited using an inhibitor (DAKO, USA) according to the manufacturer’s instructions.
The criteria to define a cell expressing MMP-2 were that of Trudel et al. (2003) [
Samples were analyzed at low power and photographed at a magnification of 400x using a digital camera, Samsung Digimax D73, and processed with the Digimax program for Windows 98. The immunocytochemical evaluation was performed by a single person, blinded to the clinical details using a coded system.
The patients were divided into 2 groups: postradical prostatectomy and bone scan negative, with or without biochemical failure, postradical prostatectomy bone scan positive with evidence of biochemical failure, defined as a PSA > 0.2 ng/mL in patients after radical prostatectomy.
Descriptive statistics were used for demographic variables, expressed as mean and standard deviation in the case of continuous variables with a normal distribution. In case of an asymmetrical distribution the median and interquartile range (IQR) values were used. Noncontiguous variables were presented as frequencies. The Student’s
The study was directed with complete conformity with the principles of the declaration of Helsinki and approval of the local ethical committees.
185 men bone scan negative and 30 men bone scan positive participated in the study. The presence of circulating prostate cells in bone marrow aspirates as well as in bone marrow of prostate cancer patients was analyzed by determining PSA protein expression. CPCs were detected in 62.7%, DTCs in 62.2%, and mM in 71.4% of patients. All men bone scan positive had CPCs, DTCs, and mM detected in 100% of the cases (Table
Demographic characteristics of the study group.
Group 1 | Group 2 | |
---|---|---|
Patient number ( |
185 | 30 |
Mean age ± SD (years) at sampling | 72.2 ± 9.0 | 76.4 ± 8.7 |
Median serum PSA (IQR) (ng/mL) at sampling | 1,32 (0,01–5,77) | 43,81 (27,72–150) |
Median Gleason score at diagnosis (IQR) | 6 (5–7) | 6 (5–7) |
Median stage at diagnosis (IQR) | 3 (2-3) | 3 (2-3) |
Median time from diagnosis (IQR) (years) | 3 (1–7) | 6 (4–9) |
% ( |
||
CPCs | 62.7% (116) | 100% (30) |
DTCs | 62.2% (115) | 100% (30) |
mM | 71.4% (132) | 100% (30) |
IQR: interquartile range, CPCs: circulating prostate cells, and DTC: disseminated tumor cells.
PSA protein expression in cells present in blood, bone marrow aspirate (BMA), and biopsy of cancer patients was compared with the Gleason score in patients without evidence of micrometastatic disease. There was no difference in the detection of cells in relation to age or serum PSA levels or the time from diagnosis to test time. Patients with higher Gleason scores had significantly higher stage disease. There were no differences in the frequency of detection of CPCs and DTCs with regards to Gleason score; however, the frequency of detection of mM was significantly lower in patients with Gleason 4 in comparison with higher Gleason scores (Kruskal-Wallis,
Demographic variables according to Gleason score.
Gleason 4 | Gleason 5 + 6 | Gleason 7 | Gleason 8 + 9 |
|
|
---|---|---|---|---|---|
No. of patients ( |
28 | 106 | 31 | 20 | |
Mean age ± SD (years) | 71.1 ± 8.7 | 73.2 ± 9.4 | 70 ± 8.9 | 71.7 ± 6.7 | NS |
Median serum PSA (IQR) ng/mL | 1.0 (0.5–4.8) | 1.68 (0.5–5.5) | 0.57 (0.1–10.0) | 1.68 (0.32–28.7) | NS |
Median stage (IQR) | 2 (1-2)a,b,c | 3 (2-3)a,d | 3 (2-3)b | 3 (3-4)c,d | a-a < 0.001 |
Median time from diagnosis (IQR) (years) | 2 (1–4) | 4 (1–8) | 3 (1–5) | 2 (1–5) | NS (Kruksal-Wallis) |
Detection prostate cells % ( |
Chi-squared | ||||
CPC | 46.4% (13) | 63.2 (67) | 64.5 (20) | 80 (16) |
|
DTC | 35.7% (10) | 65.1(69) | 67.7 (21) | 75 (15) |
|
mM | 32.1% (9) | 77.4 (82) | 77.4 (24) | 85 (17) |
|
IQR: interquartile range, CPC: circulating prostate cell, DTC: disseminated tumor cell, mM: micrometastasis, and NS: not significant.
PSA protein expression in cells present in blood, bone marrow aspirates (BMA), and biopsy of cancer patients with macrometastasis was compared with the Gleason score. There were no significant differences in the frequency of detection of CPCs, DTCs or mM with regard to Gleason score or relation to the serum PSA at the time of sampling (Table
Demographic variables according to Gleason score in men with metastastic disease.
Gleason 4 | Gleason 5 + 6 | Gleason 7 | Gleason 8 + 9 |
|
|
---|---|---|---|---|---|
No. of patients ( |
1 | 13 | 11 | 5 | |
Mean age ± SD (years) | 75.1 ± 7.7 | 74.2 ± 8.7 | 75 ± 7.2 | 74.2 ± 6.5 | NS (ANOVA) |
Median serum PSA (IQR) ng/mL | 26 | 29 (19.0–150) | 31 (22–150.0) | 30 (19–150) | NS (Kruksal-Wallis) |
Median stage at diagnosis | 3 | 3 | 3 | 3 | NS (Kruksal-Wallis) |
Median time from diagnosis (IQR) (years) | 8 | 8 (5–11) | 7 (6–9) | 7 (4–9) | NS (Kruksal-Wallis) |
IQR: inter-quartile range, CPC: circulating prostate cell, DTC: disseminated tumor cell mM: micrometastasis, and NS: not significant.
The expression of matrix metalloproteinase-2 (MMP-2), in patients positive for prostate cells in blood (
Frequency of MMP-2 expression in CPCs, DTCs, and mM in patients with nonmetastatic disease.
No. of patients | Gleason 4 | Gleason 5 + 6 | Gleason 7 | Gleason 8 + 9 |
|
---|---|---|---|---|---|
Total 100% ( |
15.1% (28) | 57.3% (106) | 16.8% (31) | 10.8% (20) | |
CPC positive |
46.4% (13) | 63.2% (67) | 64.5% (20) | 80.0% (16) | NS |
MMP-2 | 100% (13) | 100% (67) | 100% (20) | 100% (16) | NS |
DTC positive |
35.7% (10) | 65.1% (69) | 67.7% (21) | 75.0% (15) | |
MMP-2 | 100% (10) | 100% (69) | 100% (21) | 100% (15) | NS |
mM positive |
32.1% (9) | 77.4% (82) | 77.4% (22) | 85% (17) | |
MMP-2 | 0%a,b,c | 14.6%a,d (11) | 20.8%b (5) | 41.1%c,d (7) | a-a < 0.002 |
CPC: circulating prostate cell, DTC: disseminated tumor cell, mM: micrometastasis, and NS: not significant.
There was concordance in MMP-2 expression between CPCs and DTCs but not with mM for all Gleason scores in men with nonmetastatic cancer.
In men with metastatic disease MMP-2 expression was present in all CPCs and DTCs as well as mM but was expressed in all parts of the bone marrow fragment, defined as central expression (Figures
Concordance between the expression of MMP-2 in CPCs, DTCs, and mM according to Gleason score.
Kappa: MMP-2 | Gleason 4 | Gleason 5 + 6 | Gleason 7 | Gleason 8 + 9 |
---|---|---|---|---|
CPC + DTC | 0.64 | 0.59 | 0.78 | 0.57 |
CPC + mM | 0 | 0.14 | 0.19 | 0.23 |
DTC + mM | 0 | 0.13 | 0.17 | 0.30 |
Kappa values: 0–0.2 no concordance, 0.21–0.40 low concordance, 0.41–0.60 moderate concordance, 0.61–0.8 good concordance, >0.80 excellent concordance.
Stromal cell expression of MMP-2 was variable, and in the majority of microfragments MMP-2 negative the stromal cells were also negative (Figure
Expression of MMP-2 in mM and surrounding stromal cells in patients with MMP-2 expressing mM.
No. of patients | Gleason 4 | Gleason 5 + 6 | Gleason 7 | Gleason 8 + 9 | |
---|---|---|---|---|---|
mM positive 71.4% ( |
32.1% (9) | 77.4% (82) | 77.4% (22) | 85% (17) | |
MMP-2 in mM | 0% | 14.6% (11) | 20.8% (5) | 41.1% (7) | Trend chi squared |
MMP-2 in stromal cells | 0% | 0% | 4.5% (1) | 11.8% (2) |
MMP-2 is one of a family of enzymes that cleave a broad range of components of the extracellular matrix (ECM), basement membrane, growth factors, and cell surface receptors [
We believe that this is the first paper to describe the expression of MMP-2 in CPCs, DTCs, and mM. That both CPCs and DTCs express MMP-2 is consistent with the theory of the role of MMP-2 in the metastatic process of dissemination that cells expressing MMP-2 are able to penetrate the basement membrane and spread via the blood. That there is no association with the clinical parameters is in agreement with studies on prostate tissues [
There is a differential expression of MMP-2 in bone marrow micrometastasis, where the presence of MMP-2 detected by immunocytochemistry is in almost all cases zero in low grade cancer and suggests the inhibition of MMP-2. That the stromal microenvironment plays a critical role in determining tumor cell behavior has been shown in primary tumors [
That the expression in mM is different to that in CPCs and DTCs is a supportive evidence that prostate cells detected in bone marrow aspirates are different and are not true micrometastasis [
We propose that the expression of MMP-2 is inhibited by bone marrow stromal cells, in a process similar to that seen with hematopoietic stem cells, possibly by TIMP-2, although that other inhibitors modulate this function cannot be ruled out.
The inhibition of MMP-2 decreases the ability of the cancer to migrate from its new site, but does not inhibit proliferation directly. However, the decreased release of growth factors produced by MMP-2 and decreased initiation of angiogenesis by MMP-9 induced in part by MMP-2 [
In men with bone scan positive prostate cancer the expression of MMP-2 is throughout the bone fragment and involves the surrounding stromal tissue. These men had been previously treated with standard androgen blockade, and the overexpression of HER-2 protein caused by prior androgen blockade could increase MMP-2 expression and as a consequence MMP-2 is found throughout the microfragment (central pattern) and the surrounding stromal cells. Thus there may be two mechanisms involved in MMP-2 expression seen in the microfragments, firstly a passive phenomenon caused by cell proliferation towards the intertrabecular space resulting in decreased suppression of MMP-2 and secondly in more advanced disease, an active mechanism whereby HER-2 coexpression increases MMP-2 expression.
There is evidence for tumor-stroma crosstalk at metastatic sites; Kaminski et al. [
This process may have important clinical implications; firstly the differential expression of MMP-2 between circulating cells and micrometastasis could explain the early dissemination of cancer cells through mechanisms mediated for MMP-2; having invaded the bone, the inhibition of MMP-2 has a direct effect of trapping the cancer cell in its new environment and through indirect processes limits its growth in terms of the size of the focus (bone scan negative), thus could explain why although bone marrow micrometastasis are frequent, local gross recurrence is more common than metastatic relapse [
In patients with macrometastasis, that is, bone scan positive patients, these inhibitory mechanisms have been overcome, possibly by HER-2 overexpression, and MMP-2 expression is found throughout the bone marrow fragment; this in turn permits activation of the physiological mechanism previously mentioned, angiogenesis and growth of the secondary tumor.
Secondly, one of the mechanisms of action of the bisphosphonates is inhibiting MMP-2 through MMP-14 (MMP-MT1), if as we have shown that micrometastases do not express MMP-2. This may explain why clinical studies of bisphosphonates in prostate cancer patients have shown conflicting results in bone scan negative patients [
The study was funded by a research grant from the Teaching Council, Hospital de Carabineros de Chile, Santiago. There was no conflict of interests. The authors would like to thank Mrs. Ana Maria Palazuelos for her help in this study.