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Keywords:

  • colorectal cancer;
  • MMP-9;
  • metastasis;
  • disease stage

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Colorectal cancer mortality largely reflects metastasis, the spread of the disease to distant organs. Matrix metalloproteinase 9 (MMP-9) is a key regulator of metastasis and a target for anticancer strategies in colon cancer. Here, the overexpression of MMP-9 in pure tumor epithelial, but nor stromal, cell populations was associated with metastatic progression of colorectal cancer, as defined by reverse transcriptase-polymerase chain reaction (qRT-PCR) and confirmed by immunostaining. Thus, cancer cell MMP-9 represents a novel, selective prognostic and predictive factor that may be exploited for more effective disease stage stratification and therapeutic regimen selection in patients with colorectal cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Prevention or interruption of metastatic disease progression represents a therapeutic gap in the management of patients with colorectal cancer, the second leading cause of tumor-related mortality in developed nations.1,2 Metastasis results from the dissemination of select cell clones, exhibiting aberrant migratory and survival abilities, from the primary tumor to distant organs, where they establish extraintestinal colonies and remodel the local parenchyma.3,4 Uniformly, this process drastically aggravates the patient outlook, and less than 10% of patients with colon cancer metastasis at distant organs survive beyond 5 years from diagnosis.1 Effective strategies to reduce mortality and improve patient outcomes include early detection of premetastatic disease and timely recognition of metastatic spread.2 However, reliable pathological markers of metastatic colorectal cancer are scarce, as reflected by the genotypic and phenotypic heterogeneity of colon cancer cells and the variability in the analytical methods employed.3–5 Moreover, despite the progress of current chemotherapeutic regimens, advanced colorectal cancer with distant metastatic deposits remains incurable.6–8 Thus, the identification of novel, selective prognostic indicators and therapeutic targets of metastatic disease progression are of paramount importance in reducing morbidity and mortality from colorectal cancer.

Matrix metalloproteinases (MMPs) are zinc-dependent metalloendopeptidases that mediate metastasis.9,10 Cancer cells employ MMPs to cleave the extracellular matrix and invade the surrounding tissues.10,11 Specifically, in colon cancer, MMP-9 has emerged as a critical prometastatic protease regulating tumor cell growth, mobility, and survival.8,11,12 A complex interplay among MMP-9 activity, dynamic membrane regions, and signaling by adhesion molecules regulates tumor cell migration, invasion, and metastasis.13–15 Further, MMP-9 promotes tumor neovascularization by specifically activating angiogenic factors at the cancer cell/matrix interface.16 Consistent with the hypothesis that MMP-9 is a key regulator of the malignant phenotype, colorectal tumors from patients exhibit an overexpression of MMP-9 compared to matched normal adjacent tissues.8,12 In that context, stromal cells within the primary colorectal tumor are principal MMP-9 producers and may promote invasion and metastasis by cancer cells.17,18 However, human colorectal cancer cells also exhibit cell autonomous abilities to synthesize and secrete MMP-9, an effect associated with the induction of MMP-9-dependent proteolytic functions in the pericellular space that mediate metastasis.8 Indeed, tumor epithelial cell MMP-9 promotes extracellular matrix degradation, actin polymerization-driven cell spreading, and hematogenous tumor cell seeding of mouse lungs.8

In principle, tumor epithelial cell MMP-9 may represent a specific diagnostic and therapeutic target for colorectal cancer metastasis.8 The obstacles to these clinical applications include the unknown prognostic role of cancer cell MMP-9, the contribution of stroma-derived MMP-9 to cancer cell pathobiology, and the absence of approaches that selectively target MMP-9 functions mediating metastasis. Here, a previously unrecognized potential for colon tumor epithelial cell MMP-9 as a specific and selective biomarker of metastatic disease progression is revealed. Specifically, the relative increase in MMP-9 expression in tumor intestinal epithelial, but not stromal, cells compared to matched normal mucosa cells was demonstrated to predict locoregional metastatic propagation and correlate with advanced disease stages. Together, these observations suggest that MMP-9 in tumor epithelial cells is a novel prognostic and predictive marker and a potential therapeutic target for patients with colorectal cancer.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Clinical specimens

Fresh surgical specimens from 11 patients (Table 1) with histologically confirmed adenocarcinomas of the colo-rectum were obtained from the Department of Pathology, Anatomy, and Cell Biology of Thomas Jefferson University (Philadelphia, PA, USA) under a protocol approved by the Institutional Review Board.

Table 1.  Clinicopathologic parameters of colorectal cancer patients.
Age (y)
  1. *Tis, limited to mucosa (carcinoma in situ); T1, limited to submucosa; T2, invading the muscularis propria; T3, invading the serosa; T4, invading adjacent organs.

Median (range)61 (54–85)
Gender (%)
Male7 (63.6)
Female4 (36.3)
Tumor Site (%)
Colon10 (90.9)
Rectum1 (9.1)
Dukes' Tumor Stage (%)
A1 (9.1)
B3 (27.2)
C5 (45.5)
D2 (18.2)
Differentiation Grade (%)
Well1 (9.1)
Moderate7 (63.6)
Poor3 (27.2)
Tumor Depth* (%)
Tis0 (0)
T10 (0)
T21 (9.1)
T39 (81.8)
T41 (9.1)
Lymph Node Metastasis (%)
Yes7 (63.6)
No4 (36.3)
Distant Metastasis (%)
Yes2 (18.2)
Not determined9 (81.8)

Isolation of pure intestinal cell populations

Colorectal tumors and normal adjacent tissues were subjected to proteolytic digestion by incubation (37°C for 1 hour) in L-15 medium containing 10% FBS, 0.1 μg/mL insulin, 50 ng/mL epidermal growth factor (EGF), 2 mM L-glutamine, 100 μg/mL penicillin and streptomycin, 125 μg/mL fungizone and 1,250 units/mL collagenase XI. Following digestion, dissociated intestinal cells were passed through a cell strainer, magnetically labeled with magnetic microbeads conjugated to a mouse antibody directed against the human epithelial antigen (HEA), and transferred into a magnetic activated cell sorting (MACS) column (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing, to recover the unlabeled fraction, which includes stromal cells, magnetically retained HEA+ cells were removed from the magnetic field and collected with MACS elution buffer. To further purify the intestinal epithelial cells from residual cell contamination, MACS-sorted HEA+ cells were fluorescently labeled by incubation with PE-conjugated mouse anti-human CD104 (BD Biosciences, San Jose, CA, USA), another specific intestinal epithelial cell marker, and subjected to fluorescent activated cell sorting (FACS) by employing a FACS caliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The MACS flow-through (unlabeled fraction) was also subjected to FACS by employing fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (Miltenyi Biotec), a stromal cell marker, to obtain purified intestinal stromal cells.

Real-time reverse transcription-PCR

Purified cell populations from patients were pelleted and resuspended in RNAlater (Ambion, Inc., Austin, TX, USA). Total RNA was isolated with the Qiagen RNA Easy kit (Qiagen, Valencia, CA, USA) and subjected to one-step reverse transcription (RT)-PCR on a 7000 Sequence Detection System for 45 cycles (95°C, 5 minutes; 94°C, 20 minutes; 62°C, 1 minute) using the Taqman® EZ RT-PCR core reagents (Applied Biosystems, Inc., Foster City, CA, USA). MMP-9 and β-actin mRNAs were specifically detected and quantified with respective fluorescently labeled primers/probe sets (Assay on Demand; Applied Biosystems). Data were analyzed using Sequence Detection Software (Applied Biosystems) with thresholds set at 0.2. Template-negative controls were run on each PCR plate.

Immunostaining

Paraffin-embedded intestinal sections (5 μm) from clinical specimens were deparaffinized and rehydrated with sequential washes of xylene, ethanol, and water. The antigens were unmasked by heating twice at 100°C for 5 minutes in 10 mM citric buffer, pH 6.0. For immunohistochemistry (IHC), the tissue sections were incubated overnight at 4°C with a goat polyclonal antibody against human MMP-9 (R&D Systems, Minneapolis, MN, USA; 20 μg/mL), followed by incubation with the secondary antibody and diaminobenzidine (DAB) substrate (Avidin-biotin kit; Vector Laboratory, Burlingame, CA, USA). For immunofluorescence, the tissue slides were first incubated overnight at room temperature with the goat polyclonal antibody against human MMP-9 (R&D Systems; 20 μg/mL) or the rabbit polyclonal antibody against human β-catenin (Santa Cruz, Santa Cruz, CA, USA; 1:50). Then, the specific primary antibody binding was visualized by employing AlexaFluor 555-conjugated donkey anti-goat or AlexaFluor 488-conjugated donkey anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR, USA). The sections were counterstained with Slowfade Gold antifade reagent with 4′6-diamidino-2-phenylindole (DAPI) (Molecular Probes) to identify individual cells and nuclei and examined with a Zeiss LSM 510 Meta confocal microscope. The secondary antibody alone was used as negative control.

Statistical analysis

Unless otherwise indicated, data are expressed as the mean ± standard error of mean (SEM). The association between patient subgroups with relative stromal or epithelial MMP-9 overexpression and the degree of lymph node involvement was evaluated by employing the exact version of Cochran-Armitage Trend test. The differences in stage grouping scores and percentage of positive nodes between patients with increased and decreased MMP-9 mRNA levels in tumor cell populations were determined by the Wilcoxon two-sample test. Data were analyzed employing StatExact 7 (Sytel Software Corporation, Cambridge, MA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Increased MMP-9 expression in colorectal tumor cells is associated with locoregional metastatic dissemination

Normal adjacent intestinal mucosa and colorectal tumors from 11 patients were subjected to collagenase digestion. Crude populations enriched in epithelial or stromal cells were first obtained by isolating cells positive and negative for HEA from the bulk of dissociated human intestinal cells by employing MACS sorting. Purified intestinal epithelial (Figure 1, top panels) and stromal (Figure 1, bottom panels) cells were collected by employing FACS sorting with PE-conjugated anti-CD104 (on MACS-isolated HEA+ cells) and FITC-conjugated anti-CD45 (on unlabeled cells of the MACS fow-through), respectively. Then, isolated epithelial and stromal cells were subjected to real-time RT-PCR for quantification of MMP-9 mRNA. MMP-9 was expressed in all epithelial and stromal cells from patients examined (Figure 2A). Further, within the epithelial or stromal cell populations, MMP-9 expression was increased or decreased in cells purified from tumors compared to matched normal adjacent tissues (Figure 2A). Importantly, local metastasis to regional lymph nodes occurred only in patients exhibiting increased MMP-9 expression in tumor cells (Figure 2B). In contrast, all cancer patients with decreased MMP-9 levels in both epithelial and stromal cells from tumors were node-negative (Figure 2B).

image

Figure 1. Isolation of pure epithelial and stromal cell populations from primary tumors and matched normal mucosa of patients with colorectal cancer. Following collagenase digestion and MACS sorting, the epithelial (MACS elution) or stromal (MACS flow-through) cells of normal adjacent tissue (NAT) and colorectal tumors (Tumor) isolated from patients were subjected to FACS sorting. Pure intestinal epithelial (red box for PE-conjugated anti-CD104) or stromal (green box for FITC-conjugated anti-CD45) cell populations were collected for further analysis.

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image

Figure 2. Relative MMP-9 expression levels in colorectal tumor cell populations exhibit prognostic significance. (A) Stromal and epithelial cells from primary colorectal tumors (Tumor) and normal adjacent tissue (NAT) isolated by MACS and FACS sorting were subjected to RT-PCR. Sample levels of MMP-9 mRNA were normalized to respective β-actin mRNA using the formula 2[(MMP-9Ct) −(β-actinCt)], where Ct is the sample threshold cycle number. The lines connect data from matched specimens from the same patient. The box plots denote median and values from the 25th to 75th percentile; the whiskers denote range of values. (B) Lymph nodes involvement in the different subgroups of patients as defined by the relative expression of tumor cell MMP-9 compared to the matched normal mucosa cell MMP-9. [UPWARDS ARROW]MMP-9 = MMP-9 overexpression; [DOWNWARDS ARROW]MMP-9 = MMP-9 downregulation; E = tumor epithelial cells; S = tumor stromal cells.

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MMP-9 in tumor epithelial cells is a prognostic indicator of metastatic colorectal cancer

Of significance, 100% of patients (n= 5) with relative MMP-9 overexpression in tumor epithelial cells, compared to normal epithelial cells, exhibited lymph node involvement. Among the different pathologic subgroups defined by the relative MMP-9 expression in tumors, colorectal cancer patients with tumor epithelial cell MMP-9 overexpression exhibited the highest metastatic tumor burden in regional lymph nodes (Figure 2B). Conversely, only 83% of patients (n= 6) with relative MMP-9 overexpression in tumor stromal cells, compared to stromal cells from the adjacent normal mucosa, had node-positive disease, as reflected by low-to-intermediate metastatic involvement (Figure 2B). Accordingly, the proportion of patients with relative overexpression of MMP-9 in tumor epithelial, but not stromal, cells increased significantly in higher node-positive stages (N0 0%, N1 50%, N2 100%), as defined by employing the tumor node metastasis (TNM) system by the American Joint Committee on Cancer (AJCC) (Figure 3, lef panels).19 Attribution of an arbitrary score to the TNM-based stage grouping of patients examined (Table 2) revealed that patients with increased MMP-9 levels in tumor epithelial cells exhibited the most advanced stage of disease, which was significantly higher than that of patients (n= 6) with decreased MMP-9 expression in cancer cell populations (stage score: 6.60 ± 0.27 in patients with increased cancer cell MMP-9 vs. 3.83 ± 0.77 in patients with decreased cancer cell MMP-9, p= 0.022; Figure 3, bottom-middle panel). No significant difference in stage grouping scores was detected between patients with relative MMP-9 overexpression in tumor stromal cells and those with relative MMP-9 downregulation in the same cell population (Figure 3, upper-middle panel). Further, patients with a relative increase in the expression of MMP-9 mRNA in epithelial cells in tumors, compared to matched normal tissues, exhibited a significantly higher proportion of regional lymph nodes harboring metastases than patients with a relative decrease in MMP-9 expression (percentage of lymph node-positive: 27.20 ± 9.60 in patients with increased cancer cell MMP-9 vs. 4.85 ± 4.00 in patients with decreased cancer cell MMP-9, p= 0.017; Figure 3, bottom-right panel). In contrast, the disease burden in patients did not correlate with the relative changes in MMP-9 expression in tumor stromal cells compared to normal adjacent tissues (Figure 3, upper-right panel).

image

Figure 3. MMP-9 overexpression in tumor epithelial cells is a biological marker of colorectal cancer metastasis. Association between MMP-9 overexpression in tumor cell populations and lymph node involvement (left panels). Compared to those with tumor MMP-9 downregulation, patients with MMP-9 overexpression in tumor epithelial, but not stromal, cells exhibit advanced disease stage (middle panels) and increased lymph node tumor burden (right panels). N categories and disease stage scores are specified in Table 2 and are based on the AJCC/TNM system.19[UPWARDS ARROW]MMP-9 = MMP-9 overexpression; [DOWNWARDS ARROW]MMP-9 = MMP-9 downregulation. The values in the middle and right panels are means ± SEM. *p <0.05 by the Wilcoxon two-sample test.

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Table 2.  TNM-based stage group and score for colorectal cancer patients.
Stage GroupTNM*Lymph Nodes Examined (Range)Patient Number (%)Score
  1. *T categories are as described in Table 1; N0, no lymph nodes involvement; N1, metastasis in 1-3 lymph nodes; N2, metastasis in ≥4 lymph nodes; M0, no distant metastasis; M1, metastasis at distant organs.

  2. + No patients were included in the stage IV group because almost universally the M category was not determined (Table I).

0Tis, NO, MO00 (0)1
ITl-2, NO, MO271 (9.1)2
IIAT3, NO, MO13–233 (27.3)3
IIBT4, NO, MO00 (0)4
IIIATl-2, Nl, MO00 (0)5
IIIBT3-4, Nl, MO9–294 (36.3)6
IIICTl-4, N2, MO13–333 (27.3)7
IV+Tl-4, NO-2, Ml008

Overexpression of MMP-9 in colon tumor cell compartments is detected by conventional histopathology

Paraffin-embedded tissue slides from patients with colorectal cancer were immunostained for MMP-9 (Figure 4). IHC analysis revealed that MMP-9 protein is detected in both normal adjacent tissues and primary tumors from colon cancer patients (Figure 4, lef panels). Moreover, confocal microscopy of tissue specimens from a patient with node-positive disease demonstrated an overexpression of MMP-9 protein in primary tumor cell compartments, compared to cells from the matched normal adjacent mucosa (Figure 4, right panels). Importantly, MMP-9 in tumor epithelial cells was specifically identified by co-localization of immunofluorescent signals from MMP-9 and β-catenin (Figure 4, right panels). These analyses revealed that increased MMP-9 protein in colon epithelial cell populations of the primary tumor could be detected by routine histopathological techniques (Figure 4, right panels).

image

Figure 4. Overexpression of tumor epithelial cell MMP-9 in metastatic colorectal cancer is detected by immunostaining. IHC (left panels) and immunofluorescence (right panels) analyses of primary tumors (Tumor) and matched normal adjacent tissues (NAT) from colorectal cancer patients with metastatic disease progression. For IHC (magnification 40×), the tissues were stained with specific goat polyclonal anti-MMP-9 (brown) and hematoxylin (blue, nuclei). For immunofluorescence, the tissues were stained with DAPI (blue, nuclei) and specific antibodies against MMP-9 (red) and the epithelial-specific marker β-catenin (green) and subjected to confocal microscopy (magnification 100×).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

The mortality rate for large bowel cancer, approximately 50%, largely reflects metastatic disease progression, and the tumor deposits at distant locations signify unresectable and untreatable disease.1,19 Although the final stages are irreversible, formation of metastatic foci of colon cancer cells at extraintestinal sites is a vulnerable, multistep process. Thus, for hematogenous metastatic dissemination, cancer cells have to detach from the surrounding stroma, migrate through tissue boundaries and invade the primary organ, intravasate into the blood vessels, and embolize and distribute to distant tissues.4 Following tumor seeding, only select cancer cells with unique abilities to engage in productive cell–matrix interactions with the host parenchyma proliferate and form the metastatic niche.3,20 Further, to establish enduring metastasis, colon cancer cells have to evade local immune antitumor defenses, maliciously remodel the host microenvironment, and develop an autonomous vascular network.4,21 As a result, metastasis is a highly selective and ineficient process and >99.99% of the intravasated tumor cells are eliminated before reaching their final destination.22 The complexity of the metastatic process, and its attendant rate limitation associated with the disruption of any individual step, is at the basis of the efficacy of postsurgical adjuvant chemotherapy to prolong survival and reduce mortality in patients with early metastatic colorectal cancer, as determined by lymph node involvement in the absence of distant metastases.23

A current pathologic assessment of regional colorectal cancer metastasis is performed by examining the mesenteric nodes in surgical colectomy specimens with traditional hematoxylin-eosin staining.24 However, the accuracy in estimating node tumor burden is hampered by the great variability in nodal harvest, as a result of interpatient anatomic differences, the extent of surgical lymphadenectomy, and the ability to search for lymph nodes by the pathologist.25 The unclear number of lymph nodes to be analyzed and the imperfect sensitivity of histopathology represent other obstacles for an appropriate metastatic disease evaluation.24,25 Although the introduction of newer molecular-based methods of diagnosis selective for the metastatic process has been invoked,5 specific biological markers of colorectal cancer metastasis have not yet emerged. Here, node-positive disease always was associated with an increased MMP-9 expression in tumor cell compartments compared to matched normal mucosa cells, indicating that the relative increase in MMP-9 expression in colorectal tumor cells is a specific biomarker of metastatic dissemination. Importantly, MMP-9 overexpression in cells of the primary tumors could be detected by both RT-PCR and standard IHC techniques. The implications of these findings on patient management are important because they suggest that pathological analysis of the primary tumor, regardless of lymph node status, may be sufficient for detecting early colorectal cancer metastasis.

Pathologic staging of patients remains the most important prognostic factor for colorectal cancer.7 Indeed, the survival rate of these patients is inversely correlated with the AJCC/TNM stage (Table 2), and while more than 90% of patients with stage I survive 5 years from the initial diagnosis, less than 60% or 10% of them are still alive when metastasis has spread to regional (stage III) or distant (stage IV) locations, respectively.1,19 There is a considerable risk to understage patients with the current histological methods of pathologic node evaluation.5,25 Specifically, a clear prognostic dishomogeneity exists for the node-positive stage III group, and various permutations of the N category have been proposed to better stratify colorectal cancer patients with lymph node involvement.24 Here, the relative MMP-9 overexpression in tumor epithelial cells, compared to normal epithelial cells, predicted lymph node involvement and correlated with an increased metastatic tumor burden. Importantly, the patient population exhibiting a relative increase in tumor epithelial cell MMP-9 was significantly enriched in higher node-positive stages, suggesting that cancer cell MMP-9 overexpression is a novel prognostic factor in colon cancer. Moreover, colorectal cancer group staging is the fundamental predictive factor for patient management. Thus, premetastatic disease stages often require surgery alone with curative outcomes, while metastatic stages warrant postsurgical adjuvant chemotherapy with the intent to improve expectancy and quality of life.6,7 A careful stratification of the node-positive stage III groups, in turn, may represent the key discriminator for directing patients to less (for stage IIIA), intermediate (for stage IIIB), or more (for stage IIIC) aggressive therapeutic regimens.24 Patients with a relative increase in MMP-9 levels in tumor epithelial cells signified advanced disease stages, characterized by significantly higher proportion of regional lymph nodes harboring metastasis, compared to patients with a relative decrease in MMP-9 expression. Thus, MMP-9 overexpression in primary tumor epithelial cells may represent a highly specific molecular marker for both the prognostic and predictive stratification of patients with colorectal cancer.

In colorectal tumors, the fibroblasts and inflammatory cells of the stromal compartment are a principal source of MMP-9, which may confer metastatic abilities to the neighboring cancer cells.17,18 However, an examination of pure cell populations from the tumors and adjacent normal mucosa of patients with colorectal cancer demonstrated that MMP-9 of epithelial, but not stromal, cells functions as a pathological marker of metastatic disease progression. Accordingly, cancer cell MMP-9 is a critical regulator of the pathobiology underlying colorectal metastasis, selectively promoting migration, invasion, and seeding of the target organs by tumor cells.8 Thus, beyond stromal cell contribution, MMP-9 produced by tumor epithelial cells may control unique cellular functions mediating metastasis. Precise elucidations of these mechanisms underlying colorectal cancer invasion and metastasis may provide novel, highly selective, and effective therapeutic strategies for targeted prevention and treatment of colorectal cancer metastasis.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Advanced, metastatic colorectal cancer remains incurable. An accurate patient stratification into defined prognostic and predictive disease stages is essential to improve quality of life, prolong survival, and reduce mortality in colorectal cancer.5,7,25 MMP-9 overexpression in tumor epithelial cells is a novel, specific, and selective marker for the prognosis and management of patients with colorectal cancer. Further, cancer cell MMP-9 represents a novel predictive factor that may indicate treatment response and targeted therapy selection to prevent and treat metastatic disease progression. If future studies demonstrate that MMP-9 overexpression in cancer cells is an independent prognostic and predictive factor, translation of these previously undescribed MMP-9 functions into clinical practice would have the potential to profoundly impact the management and cure of patients with colorectal cancer.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

This work was supported by grants to Giovanni Mario Pitari from the Pennsylvania Department of Health. The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. Wilhelm Johannes Lubbe was supported by the NIH Institutional Training Award (5T32 CA09662).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References