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From the Department of Biochemistry and Molecular
Biology,*
Medical University of South Carolina, Charleston,
South Carolina; and the Ferring Research
Institute,
University of Southampton Research
Centre, Southampton, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another important new finding indicated that the kallikrein-kinin system stimulated angiogenesis in an in vivo model.10 In this study the femoral artery was removed in mice to induce hindlimb ischemia, and kallikrein gene delivery significantly increased capillary density and blood flow to the affected limb. This effect was reversed by bradykinin B2 and B1 receptor antagonists, demonstrating a role for both receptors in the angiogenic response. An increased expression of tissue kallikrein and bradykinin B2 receptors, as well as kinin’s promotion of growth, have previously been shown in cultured microvascular endothelial cells.11,12 In vivo, the kininogen substrate is abundant in plasma and tissues,7 so the expression and availability of kallikrein are most likely the rate-limiting factors in kinin production.
Our goal was to investigate whether tissue kallikrein possibly facilitates cancer cell invasiveness. Tissue kallikrein has been localized to neutrophils13 and has been postulated to be involved in neutrophil extravasation through the vascular endothelium and into tissues. Kinin activation of endothelial B2 receptors has been shown to cause increased intracellular calcium and subsequent endothelial retraction,14 a critical step in diapedesis. In addition to the enzymatic formation of vasoactive kinins, tissue kallikrein in vitro efficiently activates two matrix-degrading metalloproteases that are important for cancer cell mobility: progelatinase A (72-kd gelatinase, MMP-2) and progelatinase B (92-kd gelatinase, MMP-9).15,16 Our present results demonstrate that a tissue kallikrein inhibitor suppresses cancer cell invasiveness in in vitro assays and provide evidence indicating tissue kallikrein may enhance cancer cell metastasis. Tissue kallikrein inhibition may be of therapeutic value in the treatment of metastatic disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four-µm sections were cut from formalin-fixed, paraffin-imbedded archival specimens from surgical resections for pancreatic adenocarcinoma. Samples of pancreatic adenocarcinoma were taken from 18 patients after Whipple procedures. Sections had previously been histologically examined and categorized by pathologists as moderately (n = 14) to poorly (n = 4) differentiated adenocarcinoma of ductal origin. Before immunohistochemistry for kallikrein, sections were cleared, rehydrated, and antigen retrieval done by steaming sections for 20 minutes while submerged in 0.1 mol/L of citrate buffer, pH 5.0.
Antibodies for Immunohistochemistry
Rabbit anti-human tissue kallikrein antiserum was used at a dilution of 1:1200 to detect kallikrein and an equal dilution of normal rabbit sera served as negative control. The specificity of the rabbit antiserum against human tissue kallikrein has been previously published.17 To identify human cancer cells a commercially available monoclonal antibody to cytokeratin 18 was used at a dilution of 1:100 (Novocastra Laboratories, Newcastle on Tyne, UK). The manufacturer reports this monoclonal is specific to human cytokeratin, and by immunohistochemistry and Western blot is not cross-reactive with rat proteins. A monoclonal to von Willebrand factor (DAKO, Carpinteria, CA) was used at a dilution of 1:200 to identify the endothelium of lung vessels. Visualization of primary antibodies was performed using the Vectastain Elite Universal avidin-biotin-peroxidase complex kit as directed by the manufacturer (Vector Laboratories, Burlingame, CA).
For the quantification of interstitial breast cancer cells in rat lungs after the explanted lung invasion assay a double-label immunohistochemistry was performed with the anti-cytokeratin 18 monoclonal used as the first primary antibody, followed by color development with diaminobenzidine tetrahydrochloride plus 1% nickel chloride to darken the chromogenic reaction product to brownish black. After incubating sections in 3% hydrogen peroxide to irreversibly inhibit the peroxidase bound to anti-cytokeratin, the antibody to von Willebrand factor was used as a second primary antibody in the same sections and subsequent development was done with 3-amino-9-ethylcarbazole to produce a red stain. A similar double label was performed with the anti-tissue kallikrein sera as the first primary antibody and anti-cytokeratin 18 as the second primary to co-localize the human-specific marker with tissue kallikrein expressed in human breast cancer cells invading rat lung during the ex vivo assay.
In Situ Hybridization Histochemistry in Tissue Sections of Pancreatic Adenocarcinoma
In situ hybridizations specific for human tissue kallikrein were performed as previously reported.18,19 A 186-bp human tissue kallikrein cDNA fragment was used to generate digoxygenin-UTP labeled antisense and sense riboprobes. Antisense riboprobe specific for tissue kallikrein was used to identify the kallikrein mRNA. Control sections were incubated with a labeled sense riboprobe. Additional controls were pretreated with RNase A before incubating with an antisense riboprobe.
Cell Line and Reagents
The human breast cancer cell line MDA-MB-231 (ATCC, Rockville, MD) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus antibiotic (penicillin 100 U/ml, streptomycin 100 µg/ml) and 10% fetal bovine serum. MDA-MB-231 was chosen for assays because this cell line metastasizes aggressively and has demonstrated high constitutive expression of progelatinases A and B (MMP-2 and MMP-9).20 MDA-MB-231 cells are estrogen receptor-negative and hormone-independent. The synthesis, characterization, and in vivo use of the peptide-based human tissue kallikrein inhibitor FE999024 (Ferring Research, Southampton, UK), formerly designated CH2856, has been previously described.21 FE999024 has a Ki of 2.2 nmol/L toward tissue kallikrein and displayed high selectivity for tissue kallikrein (potency ratios in parentheses) over plasma kallikrein (454), trypsin (454), thrombin (16,000), and plasmin (4900).
Enzyme-Linked Immunosorbent Assay for Human Tissue Kallikrein
Levels of human tissue kallikrein in MDA-MB-231 culture media and cell lysates were determined by enzyme-linked immunosorbent assay as previously described.22
Matrigel Invasion Assay
Matrigel matrix (Becton Dickinson Labware, Franklin Lakes, NJ) was applied and polymerized in 24-well 9-mm inserts containing polyethylene terephtphalate (PET) membranes with 8-µm pores to create invasion chambers as directed by the supplier (Becton Dickinson). MDA-MB-231 cells were grown to near confluence, harvested by trypsinization, which was inactivated with media containing bovine calf serum, and cells were subsequently washed twice in DMEM without added serum or proteinase inhibitor. The cells were suspended in DMEM at 1 x 105/ml. DMEM (0.6 ml) containing 5% fetal bovine serum was added to each plate well as a chemoattractant, and 0.2 ml (2 x 104 cells) of cell suspension was added to each insert. Assays were performed with triplicate wells for each condition. In experimental groups the tissue kallikrein inhibitor FE999024 was used at concentrations of 0.05, 0.5, and 5 µmol/L. Cells treated with the inhibitor were preincubated 20 minutes before applying to chambers. The plates of inserts were incubated for 6 hours at 37°C. Noncoated membrane inserts were also seeded to serve as controls. After incubation the chambers were processed and stained as directed by the supplier (Becton Dickinson). The cells were enumerated by counting four fields per chamber under x100 magnification with the aid of a ruled grid. Data were expressed as percent invasion, ie, the ratio of cells invading through the Matrigel-coated inserts relative to the uncoated control inserts.
Explanted Rodent Lung Invasion Assay
Sprague-Dawley rats weighing between 225 and 275 g (Sprague-Dawley Harlan, Indianapolis, IN) were anesthetized by an intraperitoneal injection of ketamine (9 mg/100g body weight)/xylazine (1 mg/100g body weight). The procedure was performed aseptically and all solutions contained penicillin 100 U/ml and streptomycin 100 µg/ml. The trachea was exposed and a 0.05 x 0.09-inch tygone tube inserted and placed above the bifurcation, secured by ligation, and connected to a model 683 rodent respirator (Harvard Instruments, South Nautik, MA) set at a volume of 1.0 ml of room air and a rate of 60/minute. The chest was opened and the rat heparizined by injection of 300 U of heparin into the left ventricle. The heart and lungs were carefully removed as a single block and placed in sterile phosphate-buffered saline (PBS). The pulmonary artery and left atrium were then catheterized with 0.04 x 0.07-inch tygone tubing and the pulmonary circulation perfused with 50 ml of DMEM entering the pulmonary artery by gravity flow and draining from the left atrium. Bronchial alveolar lavage was accomplished by gently instilling 5 ml of DMEM per lavage through the tracheal tube with a needle small enough to allow backflow through the endotracheal tube. The lungs were manipulated gently and inverted to drain. A total of 30 ml of DMEM was used for lavage. At the beginning of the assay 5 ml of DMEM plus 5% fetal bovine serum was instilled as in lavage to wet the airspace with chemoattractant. Five ml of DMEM with 10% bovine albumin containing 1.0 x 105 MDA-MB-231 cells, prepared the same as for invasion assays, were slowly perfused through the pulmonary artery, Lungs were then placed in DMEM maintained at 37°C, and incubated 6 hours while respirated.
Cells within the airspace were recovered by lavage performed with a total of 30 ml of sterile PBS. After airspace recovery, cells were recovered from the vasculature by perfusion of 30 ml of PBS through the left atrium and draining from the pulmonary artery and trachea. Cells were centrifuged and resuspended in 2 ml of PBS, an aliquot was diluted 1:1 with 0.2% trypan blue solution and counted in a hemocytometer. Cells were distinguishable as either large or small in size, and only large cells were counted. Microscopic examination after hematoxylin and eosin (H&E) staining confirmed large cells as morphologically anaplastic and cancerous. Small cells, ie, red blood cells or occasional leukocytes, were ignored. An invasion index was expressed as a percentage and calculated as the number of cells recovered from the airspace divided by the total cells recovered from the airspace and vessels. Recovered cancer cells were >95% viable. Five ml of zinc formalin was instilled into lungs to inflate and fix before submerging lungs completely in fixative for subsequent embedding in paraffin.
As a second index of invasion paraffin sections underwent a double-label immunohistochemistry as described above with antibodies to cytokeratin 18 and von Willebrand factor (factor VIII). The darkly stained human breast adenocarcinoma cells were counted with the aid of a counting grid in 20 random x100 fields per animal and the interstitial localization of these cells was verified at x400. Endothelial visualization helped to assure cells lodged in capillaries were not counted as invading cells. Invasion index was expressed as cytokeratin 18-positive cells embedded in interstitial matrix per mm2. Cells were also qualitatively evaluated in sections in which cytokeratin 18 immunohistochemistry was followed by picro-sirius red staining to visualize cancer cells embedded in collagen matrix. As an additional control rat hepatocytes were harvested by collagenase digestion and were washed twice in DMEM to remove enzyme and fetal bovine serum. Hepatocytes were resuspended at 1.0 x 105 cells/ml, perfused, incubated, recovered, and quantified in the same manner as breast cancer cells. Visualization of hepatocytes in lung sections was accomplished by periodic acid-Schiff (PAS) staining to demonstrate abundant cytoplasmic glycogen.
Statistical Analysis
Results are expressed as mean ± SEM. Comparisons among groups were made by analysis of variance with Fisher’s PLSD (protected least significant difference). Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
illustrates the
immunohistochemical reactivity for human tissue kallikrein typical in
pancreatic ductal adenocarcinoma both within the pancreas and at sites
of metastasis. Adenocarcinoma samples from 14 of 18 patients with
pancreatic cancer were positive for kallikrein. All four samples of
poorly differentiated adenocarcinoma were positive. Metastatic cancers
in lymph nodes were examined from eight patients (16 nodes), and in
samples from six patients they were positive for kallikrein (10
nodes). Low-power magnification typically revealed an overall
heterogeneous distribution at the sites of cancer with the most
frequent staining occurring apically. However, basolateral staining, as
indicated by the smaller arrows in Figure 1, A and C
, was also evident.
Cells morphologically identified as neutrophils, lymphocytes, and
fibroblasts were frequently positive for tissue kallikrein in inflamed
tissue adjacent to adenocarcinoma. The larger arrows in Figure 1, A and B
, point to an area of inflammatory infiltrate that was positive for
kallikrein. High-power magnification revealed a distinct, finely
granular cytoplasmic reactivity. No reactivity was observed when normal
rabbit serum was used as a negative control (not shown). H&E staining
demonstrates morphological features, such as atypical malignant glands
lined with anaplastic cells, consistent with pancreatic adenocarcinoma
of ductal origin.
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shows the in situ
localization of tissue kallikrein mRNA using specific
digoxygenin-labeled antisense riboprobes. These results indicated that
tissue kallikrein mRNA is synthesized by pancreatic adenocarcinoma
cells within the pancreas as shown in Figure 2
. By in situ
hybridization, seven samples were positive for kallikrein mRNA,
including two samples of adenocarcinoma that had metastasized to lymph
nodes. Reactivity was absent when sense riboprobe was hybridized as a
negative control. Also, no staining was seen when sections were treated
with RNase A before hybridization with the antisense probe (not shown).
These results demonstrate the specificity of labeled antisense probe
for kallikrein transcript.
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Figure 3
demonstrates the inhibition
of breast cancer cell invasion of Matrigel matrix when the human tissue
kallikrein inhibitor FE999024 was used at doses of 0.05, 0.5, and 5
µmol/L. Compared to untreated breast cancer cells, the kallikrein
inhibitor maximally suppressed invasion by 39%
(P < 0.001 as compared to untreated control) in
a dose-dependent manner. MDA-MB-231 breast cancer cells in culture
secreted 8 ng of human tissue kallikrein per ml media and had 17 ng of
kallikrein per mg of cellular protein as determined by a specific
enzyme-linked immunosorbent assay.
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Figure 4
shows the inhibition of
invasion of MDA-MB-231 breast cancer cells in the explanted lung
invasion assay as quantified after recovery of breast cancer cells by
lavage (Figure 4A)
or identification of breast cancer cells in the
interstitium by immunohistochemistry for human cytokeratin 18 (Figure 4B)
. The results are obtained during a time course of 1, 2, 4, and 6
hours after infusion of MDA-MB-231 cells into the pulmonary
circulation. The presence of 5 µmol/L of tissue kallikrein inhibitor
resulted in a 33% reduction in breast cancer cell invasion into the
airspace as quantified by lavage (graph A, 6 hours, P =
0.002), and a 34% reduction of invading cells in the lung interstitium
as quantified by cytokeratin immunohistochemistry (graph B, 6 hours,
P = 0.02).
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shows results in sections of the
explanted rodent lung invasion assay after four histochemical
procedures. Figure 5A
shows an invading breast cancer cell (arrow)
identified by anti-cytokeratin 18 immunostaining (black). The red
staining for factor VIII localizes vascular endothelium. The
double-label procedure was used so that during quantification, cancer
cells entrapped in capillaries are not counted as interstitial. Breast
cancer cells invading the interstitium were usually seen in close
proximity to capillaries, postcapillary venules, or occasionally small
arterioles. Only breast cancer cells embedded in interstitial matrix,
such as the one shown in Figure 5A
, were counted for quantification
(Figure 4B)
.
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shows the co-localization of immunoreactive tissue kallikrein
with cytokeratin 18 as demonstrated by a double-immunostain procedure.
Tissue kallikrein reactivity in breast cancer cells appears black and
immunostaining for human cytokeratin 18 appears red. The results show
tissue kallikrein localizes to human breast cancer cells during the
explanted lung invasion assay.
In the H&E-stained section of rat lung at 6 hours after infusion
(Figure 5C)
, the cancer cells in the lung interstitium (arrows) are
morphologically identified by their large size relative to lung
epithelial cells and their large, irregular nuclei. Six hours after
infusion, the respiratory epithelium and vascular endothelium appeared
intact and healthy morphologically, except in large vessels and airways
where some sloughing of cells was seen. Small areas of the lung also
appeared to have cells in the lung parenchyma with condensed nuclei,
however, the lung extracellular matrix appeared intact. These areas
were not used for quantification of interstitial invading cells. These
observations indicated that 6 hours represent approaches the maximal
time during which the assay can be conducted.
PAS staining was used to identify hepatocytes (large arrow, Figure 5D
)
that were used as noninvasive control cells. Hepatocytes stained by PAS
are identifiable by an intensely positive cytoplasm because of their
abundant intracellular glycogen. Hepatocytes were not seen in the lung
interstitium, although occasional hepatocytes were seen in alveoli,
indicating that some leakage was occurring from vessels. By lavage
recovery at 4 hours after infusion, 4.2% of hepatocytes were in the
airspace and this number increased to 5.8% by 6 hours after infusion.
Therefore, the invasion assay must be performed within 6 hours after
infusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The explanted lung assay differs qualitatively from Matrigel-coated chambers in that there is extravasation through the vascular endothelium followed by invasion into a natural extracellular matrix. This assay is more closely related to in vivo metastasis. However, problems did arise in this assay that indicated that a significant level of vascular leakage unrelated to invasion occurs in a time-dependent manner. An increasing number of hepatocytes used as noninvasive control cells were recovered from the airspace and by 6 hours after infusion, swelling of the lungs became apparent and fluid escaped through trachea during vascular recovery. Also at 6 hours after infusion, histological changes, such as sloughing of the endothelium or epithelium, were seen in the large vessels and airways. These observations indicate 6 hours is the maximum time in which the explanted lung invasion assay can be performed as detailed. The smaller vessels, which are the sites of extravasation, appeared morphologically healthy and intact at 6 hours. This is probably because of adequate aeration of the smaller vessels and hypoxia in larger vessels. However, the walls of larger vessels and airways remained intact and for our purposes of examining invasion, the 6-hour assay as performed proved adequate. The explanted lung invasion assay should be generally useful for assaying extravasation and invasion by not only cancer cells, but for neutrophils and other inflammatory cells as well. Neutrophils and other cells found in inflammatory infiltrates can be easily recovered from the pulmonary airspace and vasculature. Additionally, the short time period required for assaying neutrophils and leukocytes should facilitate interaction with a fully functional endothelium.
It is becoming apparent that a significant number of adenocarcinomas express kallikrein. These tumors likely take advantage of kallikrein’s enzymatic properties to influence growth and metastasis. Our results demonstrate the expression and localization of kallikrein in pancreatic adenocarcinoma of ductal origin. A direct comparison with normal pancreatic tissue in our samples proved difficult because pancreatitis was evident in most of the uninvolved pancreas adjacent to adenocarcinoma in our samples. However, the reactivity of morphologically normal pancreas that was present (results not shown) were consistent with earlier studies establishing the expression and localization of tissue kallikrein in acinar cells.22,26,27 Immunoreactive kallikrein and kallikrein mRNA are not normally present in the ductal epithelium. Our results suggest a possible differential expression in adenocarcinoma derived from ductal epithelium. Not all samples of adenocarcinoma examined were positive for immunoreactive kallikrein, and detection of kallikrein mRNA proved more problematic than the protein. This is probably because of the low level of expression usually seen for proteinases such as kallikrein, coupled with the variations in fixation and processing used on these archival specimens.
Our study supports the hypothesis that tissue kallikrein may contribute to extracellular matrix degradation related not only to metastasis, but potentially during tissue remodeling and repair as well. The observation that kallikrein localizes to inflammatory cells and fibroblasts is consistent with a possible contribution to matrix degradation during inflammation and repair. Potential roles for kallikrein in cancer may also include the processing of novel substrates such as growth factors. For example, kallikrein has been shown to process somatostatin in vitro.28 Recent studies, mostly related to cancer, have also revealed that novel members of the human kallikrein family exist,29,30 although their functions remain to be elucidated. The accumulating body of evidence implicating roles for tissue kallikrein and novel members of the kallikrein family in tumor biology certainly warrants confirmation and further investigation. Because the growth and mobility of many soft tissue cancers, including adenocarcinoma, use common proteinases, inhibition of these proteinases may be beneficial for the treatment of these cancers. Combination therapies incorporating proteinase inhibitors may be particularly useful, and may even prove to be effective alternatives to the less specific and much more toxic conventional chemotherapies.
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Footnotes |
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Supported by National Institutes of Health grants HL 44083 and HL 29397.
Accepted for publication July 27, 2001.
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