Human neutrophils facilitate tumor cell transendothelial migration

Qiong Di Wu1, Jiang Huai Wang1, Claire Condron2, David Bouchier-Hayes2, and H. Paul Redmond1

Department of Surgery, 1 Cork University Hospital, University College Cork, Cork, and 2 Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

metastasis; endothelial cells; adhesion molecules; cell proliferation; apoptosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DESPITE THE ADVANCES in primary tumor management, up to 50% of patients will ultimately die of their disease. Metastases from primary tumors remain a major cause of cancer-related deaths. To metastasize, tumor cells must shed into the blood stream (intravasation) directly by invasion into the tumor-derived vasculature or indirectly by lymphatic drainage, survive in the circulation, and finally migrate through normal vascular endothelium and proliferate in the target organs (extravasation). Tumor cell extravasation plays a key role in tumor metastasis. Several hypotheses have been proposed to explain tumor cell extravasation (5, 7, 11). However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain controversial.

The immune system acts to eliminate tumor cells. However, under certain circumstances, leukocytes may enhance the ability of weakly metastatic tumor cells to metastasize, or tumor cells can exploit leukocyte function to increase their metastatic efficiency (1, 22). Human polymorphonuclear neutrophils (PMN), which comprise 50-70% of circulating leukocytes, can be spontaneously cytotoxic to tumor cells. However, PMN may function to promote tumor growth and metastasis rather than to cause tumor cytolysis. For example, evidence from animal studies has shown that, in vitro, PMN increases tumor cell attachment to endothelial monolayers (21) and that tumor-elicited PMN, in contrast to normal PMN, enhances metastatic potential and invasiveness of tumor cells in an in vivo tumor-bearing rat model (24). Furthermore, under light and electron microscopes, circulating PMN have been seen in close association with metastatic tumor cells throughout the process of tumor cell arrest and extravasation in vivo (4).

Human PMN actively participate in the inflammatory response by transendothelial migration. The process of PMN transmigration through vascular endothelium can be divided into three steps: PMN rolling, adhesion, and migration. Each step is mediated by different adhesion molecules (6, 17). PMN rolling is mediated by L-selectin expressed on PMN and by E-selectin and P-selectin expressed on endothelial cells. More importantly, both PMN adhesion and migration require the expression of CD11a/CD18 (known as LFA-1) and CD11b/CD18 (known as MAC-1) on PMN and the expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells. However, it is unknown whether PMN transendothelial migration can be employed by tumor cells to assist their extravasation during the process of metastasis.

In this study we test the hypotheses that normal human PMN function is altered by tumor cells and that these PMN can assist tumor cell migration through the endothelial barrier. Using an in vitro transendothelial migration model, we demonstrate that the metastatic human breast tumor cell line MDA-MB-231 cells fail to transmigrate spontaneously. However, tumor-conditioned medium (TCM)-treated human PMN with resultant suppressed cytocidal function, delayed apoptosis, and increased adhesion receptor expression facilitate the MDA-MB-231 cells to cross normal human macro- and microvascular endothelial monolayers. These results suggest that tumor cells can exploit PMN function to enhance their metastatic potential at the step of extravasation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and monoclonal antibodies. DMEM, M199 medium, L-15 medium, Hanks' balanced salt solution (HBSS), PBS without Ca2+ and Mg2+, FCS, penicillin, streptomycin sulfate, fungizone, glutamine, and 0.05% trypsin-0.02% EDTA solution were purchased from GIBCO BRL (Paisley, UK). Endothelial cell growth supplement, 2% gelatin, D-glucose, Dextran, Tris, EDTA, sodium citrate, Triton X-100, propidium iodide (PI), FITC-conjugated dextran (70 kDa), and heparin were purchased from Sigma (St. Louis, MO). Collagenase (type I) and Ficoll were obtained from Worthington (Freehold, NJ) and Pharmacia (Uppsala, Sweden), respectively. Mouse anti-human CD11a, CD11b, CD18, CD29, CD61, and ICAM-1 monoclonal antibodies (MAbs) were purchased from Becton Dickinson (Mountain View, CA), Chemicon (Temecula, CA), and Serotec (Oxford, UK). Different integrin and ICAM-1-blocking MAbs 4B4, ICRF44, TS1/18, and P2A4 were purchased from Coulter Clone (Miami, FL), Endogen (Woburn, MA), and Chemicon, respectively.

Endothelial cell culture. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase treatment of umbilical vein and cultured on 2% gelatin-coated culture flasks (Falcon, Lincoln Park, NJ) in complete M199 medium supplemented with 20% FCS, penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), fungizone (0.25 µg/ml), heparin (16 U/ml), endothelial cell growth supplement (75 µg/ml), and 2.0 mM glutamine as previously described (10). Cells were grown at 37°C in a humidified 5% CO2 condition and subcultured by trypsinization with 0.05% trypsin-0.02% EDTA when confluent monolayers were reached. Using the immunofluorescence technique, we identified endothelial cells by typical phase contrast "cobblestone" morphology and by the presence of von Willebrand factor antigen. HUVEC were used between passages 3 and 5.

Human dermal microvascular endothelial cells (HMVEC) at passage 3 were purchased from Clonetics (San Diego, CA) and cultured in endothelial cell basal medium (Clonetics) supplemented with Bulletkit (Clonetics) including 5% FCS, human epidermal growth factor (10 ng/ml), bovine brain extract (12 µg/ml), hydrocortisone (1.0 µg/ml), gentamycin (50 µg/ml), and amphotericin B (50 ng/ml). Cells were grown at 37°C in a humidified 5% CO2 condition and split once a week. HMVEC were used until passage 8.

Isolation of human PMN. Using the dextran-Ficoll technique as described previously (23), we isolated human PMN from lithium-heparin anticoagulated blood collected from healthy adult volunteers. Isolated PMN were resuspended in complete DMEM containing 10% FCS, penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), and 2.0 mM glutamine. PMN purity was >98% with 99% viability as determined by trypan blue exclusion.

Breast tumor cell line and preparation of TCM. The metastatic human breast adenocarcinoma cell line MDA-MB-231, isolated from a pleural effusion, was obtained from the American Type Culture Collection (ATCC, Rockville, MD). MDA-MB-231 cells were grown in L-15 medium supplemented with 10% FCS, penicillin (100 units/ml), streptomycin sulfate (100 µg/ml), and 2.0 mM glutamine. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere and subcultured by trypsinization with 0.05% trypsin-0.02% EDTA when cells became confluent.

TCM from MDA-MB-231 cells was prepared as follows. MDA-MB-231 cells were grown to subconfluency (~80%) in culture medium. After being washed three times with HBSS, cells were incubated in fresh culture medium at 37°C in 5% CO2 conditions for 24 h. TCM was then harvested, centrifuged at 700 g and 4°C for 20 min, passed through 0.22-µm pore-size filters (Gelman Sciences, Ann Arbor, MI), and stored at -20°C until use.

Assessment of neutrophil and MDA-MB-231 cell migration. HUVEC and HMVEC in 0.5 ml of complete culture medium (4 × 105 cells/ml) were grown in the upper chamber of a 12-mm-diameter, collagen-coated polytetrafluoroethylene membrane Transwell (Costar, Cambridge, MA), with 1 ml of complete culture medium in the lower chamber at 37°C in a humidified 5% CO2 atmosphere for 30 h until confluent monolayers were reached. The upper and lower chambers of the Transwells were then washed twice with HBSS.

For the PMN-assisted MDA-MB-231 cell migration assay, isolated PMN (1 × 106 cells/ml) were incubated with either TCM or culture medium for 60 min. TCM-treated or medium-treated PMN (5 × 104 cells) were mixed with MDA-MB-231 cells (2.5 × 104 cells) at a 2:1 ratio in a total volume of 0.5 ml and added to the upper chamber above the endothelial cell monolayers and collagen-coated polytetrafluoroethylene membrane. One milliliter of fresh complete culture medium with or without interleukin-8 (IL-8) (75 ng/ml, final concentration) was added to the lower chamber. After incubation at 37°C in a humidified 5% CO2 atmosphere for different time points, migrated tumor cells and PMN in the lower chamber were collected and counted according to their morphological differences. In some experiments, PMN and tumor cells were pretreated with saturating concentrations of different blocking MAbs for 30 min to identify the involvement of individual integrin and ICAM-1 receptor in PMN-mediated tumor cell migration.

Assessment of endothelial monolayer permeability. The upper chamber and the lower chamber of the Transwell were washed three times with HBSS to remove unattached and transmigrated cells 24 h after PMN-mediated MDA-MB-231 tumor cell migration. FITC-conjugated dextran was then administered to the upper chamber at a concentration of 1 mg/ml. The medium of the upper and lower chambers was aspirated separately after 4 h of incubation at 37°C in a humidified 5% CO2 atmosphere. The fluorescence activity was measured under excitation at 490 nm and emission at 530 nm. Pure FITC-dextran was used to produced a standard reference curve. A permeability index was calculated according to the following formula: permeability index = [(experimental permeability - spontaneous permeability)/(membrane permeability - spontaneous permeability)] × 100%

Measurement of human PMN respiratory burst and phagocytosis. PMN respiratory burst was assessed using a BURSTTEST (Orpegen, Heidelberg, Germany). Briefly, 20 µl of the fluorogenic substrate dihydrorhodamin 123 was added to 100 µl of PMN suspension (1 × 106 cells/ml) and incubated at 37°C for 10 min. After centrifugation, the cell pellets were resuspended in 100 µl of DNA-staining solution and incubated at 4°C for 15 min. A PHAGOTEST (Orpegen) was used to determine PMN phagocytosis. Briefly, 20 µl of stabilized and opsonized FITC-labeled Escherichia coli suspension was added to 100 µl of PMN suspension (1 × 106 cells/ml) and incubated at 37°C for 10 min. After the addition of 100 µl of quenching solution, the sample was washed twice with 3 ml washing solution in an ice-water bath. After centrifugation, 100 µl of DNA-staining solution were added to cell pellets and incubated for 15 min in an ice-water bath. The analysis of PMN respiratory burst and phagocytosis was performed on a FACScan flow cytometer (Becton Dickinson). The mean channel fluorescence was detected with FL1 using logarithmic amplification on the basis of a minimum number of 10,000 cells collected and analyzed with the software Lysis II.

Fluorescence-activated cell-sorter analysis of immunofluorescence. The expression of CD11a, CD11b, and CD18 on human PMN and the expression of beta 1-, beta 2-, and beta 3-integrins and ICAM-1 on MDA-MB-231 cells were assessed by the addition of 20 µl of FITC-conjugated anti-LFA-1alpha (anti-CD11a), phycoerythrin (PE)-conjugated anti-Leu-15 (anti-CD11b), FITC-conjugated anti-LFA-1beta (anti-CD18), PE-conjugated anti-CD29 (anti-beta 1), FITC-conjugated anti-CD61 (anti-beta 3), and PE-conjugated anti-Leu-54 (anti-ICAM-1) MAbs to 100 µl of PMN or MDA-MB-231 cell suspension (1 × 106 cells/ml). FITC- and PE-conjugated isotype IgG1 and IgG2a MAbs were used as negative control. After incubation at 4°C for 30 min, beta 1-, beta 2-, and beta 3-integrins and ICAM-1 expression on PMN and MDA-MB-231 cells were analyzed on a FACScan flow cytometer for detecting the log of the mean channel fluorescence intensity with an acquisition of FL1 and FL2, respectively. The minimum number of 10,000 events was collected and analyzed with the software Lysis II.

Assessment of apoptosis. TCM-treated, culture medium-treated, and migrated PMN were incubated in 17 × 100-mm polypropylene tubes (Falcon) at 37°C in a humidified 5% CO2 atmosphere for different times. PMN apoptosis was assessed according to the percentage of cells with hypodiploid DNA using the PI staining technique (13). Briefly, after centrifugation, 1 × 106 cells were gently resuspended in 1 ml of hypotonic fluorochrome solution (50 µg/ml PI, 3.4 mM sodium citrate, 1.0 mM Tris, 0.1 mM EDTA, and 0.1% Triton X-100) and incubated in the dark at 4°C for 2 h before they were analyzed with a FACScan flow cytometer (Becton Dickinson). The forward scatter and side scatter of cell particles were simultaneously measured. The PI fluorescence of individual nuclei with an acquisition of FL2 was plotted against forward scatter, and the data were registered on a logarithmic scale. The minimum number of 5,000 events was collected and analyzed with the software Lysis II. Apoptotic cell nuclei were distinguished by their hypodiploid DNA content from the diploid DNA content of normal cell nuclei. Cell debris was excluded from analysis by raising the forward threshold. All measurements were performed under the same instrument settings.

Determination of cell proliferation. Cell proliferation was determined using 5-bromo-2'-deoxy-uridine (BrdU) labeling and detection ELISA kit (Boehringer Mannheim, Mannheim, Germany). Briefly, 100 µl of control and migrated MDA-MB-231 cell suspension (7.5 × 104 cells/ml) were seeded in 96-well plates (Costar) and incubated at 37°C in a humidified 5% CO2 atmosphere for 24 h. BrdU (10 µM) was added to culture medium and incorporated into freshly synthesized DNA. Fixed cells with DNA partially digested by nucleases were incubated with anti-BrdU MAb conjugated with peroxidase to detect incorporated BrdU. The absorbance of the colored reaction product, which is directly correlated to the level of BrdU incorporated into cellular DNA, was determined on a Microtiter Plate Reader (Dynex Technologies, Chantilly, VA).

Statistical analysis. All data are presented as the means ± SD. Statistical analysis was performed using ANOVA. Differences were judged statistically significant when the P value was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Promotion of tumor cell transendothelial migration by TCM-treated PMN. MDA-MB-231 cells at different concentrations (2.5 × 104 and 7.5 × 104 cells) were cocultured with endothelial cell monolayers to assess tumor cell spontaneous migration. As shown in Fig. 1, A and B, tumor cells themselves did not migrate spontaneously through HUVEC and HMVEC monolayers. Human PMN incubated with culture medium failed to assist tumor cell transendothelial migration. However, PMN treated with TCM significantly promoted MDA-MB-231 cells crossing macro- and microvascular endothelial monolayers after 6 h of incubation (10.9 ± 2.8 and 12.8 ± 2.7% of tumor cell migration, respectively; P < 0.05 vs. PMN incubated with culture medium). Figure 2 shows migrated MDA-MB-231 cells after TCM-treated PMN coculture and the attachment of PMN to tumor cells. Maximum tumor cell migration occurred at 6 h in a 24-h period of incubation, whereas PMN treated with medium alone had no effect on tumor cell migration (Fig. 3).


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Fig. 1.   Tumor-conditioned medium (TCM)-treated human polymorphonuclear neutrophils (PMN) facilitate tumor cell transendothelial migration. MDA-MB-231 cell transmigration through human umbilical vein endothelial cell (HUVEC) (A) and human dermal microvascular endothelial cell (HMVEC) (B) monolayers after 6 h of incubation was assessed as described in MATERIALS AND METHODS. MDA (1) and MDA (2) represent 2.5 × 104 and 7.5 × 104 MDA-MB-231 cells. Data are expressed as the means ± SD and are representative of 5 separate experiments. The statistical significance was compared with culture medium-treated PMN, *P < 0.05.



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Fig. 2.   Migrated MDA-MB-231 cells and human PMN stained using Diff-Quick. Large cells are MDA-MB-231 cells, and cells with polymorphonuclears are human PMN. Note the attachment of PMN to tumor cells.



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Fig. 3.   The effect of TCM-treated or culture medium-treated human PMN on tumor cell transmigration. MDA-MB-231 cell migration at different time points was assessed as described in MATERIALS AND METHODS. The results are expressed as the means ± SD from 5 separate experiments.

To investigate whether TCM-treated PMN could damage the endothelial monolayer, we performed an endothelial monolayer permeability assay. As shown in Fig. 4, TCM-treated PMN did not cause a significant increase in endothelial monolayer permeability compared with culture medium-treated PMN, although the permeability index in both culture medium-treated PMN and TCM-treated PMN groups was significantly higher than in MDA-MB-231 cell alone groups.


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Fig. 4.   Endothelial monolayer permeability 24 h after human PMN and MDA-MB-231 cell transmigration. Permeability index after 4 h of incubation of FITC-conjugated dextran was assessed as described in MATERIALS AND METHODS. MDA (1) and MDA (2) represent 2.5 × 104 and 7.5 × 104 MDA-MB-231 cells. Data are expressed as the means ± SD from 4 separate experiments. The statistical significance was compared with MDA-MB-231 cell alone, *P < 0.05.

IL-8 is a potent chemotaxin and has been found to increase PMN transmigration both in vitro and in vivo (20). Therefore, we also assessed the effect of IL-8 on PMN-induced tumor cell migration. IL-8 at 75 ng/ml significantly enhanced MDA-MB-231 cell migration in TCM-treated PMN group (Fig. 5). However, IL-8 did not increase MDA-MB-231 cell migration in culture medium-treated PMN group, although IL-8 significantly increased PMN themselves transmigration (data not shown).


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Fig. 5.   The effect of interleukin-8 (IL-8) on TCM-treated or culture medium-treated PMN-induced tumor cell migration. MDA-MB-231 cell transmigration through endothelial monolayers following 6 h of incubation was assessed as described in MATERIALS AND METHODS. Data are expressed as the means ± SD and are representative of 4 separate experiments. The statistical significance was compared with culture medium-treated PMN, *P < 0.05, and TCM-treated PMN without IL-8, @P < 0.05.

In vitro viability and proliferation of migrated tumor cells. To evaluate the proliferative potential of migrated tumor cells, we assessed both viability and proliferation of migrated MDA-MB-231 cells collected after 6 h incubation. As shown in Fig. 6A, there was no significant difference in viability between migrated and control tumor cells (96 ± 6 vs. 98 ± 4%). Furthermore, the process of TCM-treated, PMN-induced tumor cell transmigration did not impair the potential of tumor cell proliferation, as migrated MDA-MB-231 cells still possessed 106 ± 8% proliferation rate compared with the proliferation rate in control tumor cells (Fig. 6B).


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Fig. 6.   MDA-MB-231 cell viability (A) and proliferation (B) before and after transendothelial migration. Cell viability was determined by trypan blue exclusion, and cell proliferation was measured as described in MATERIALS AND METHODS. Data are expressed as the means ± SD from 5 separate experiments.

Expression of beta -integrins and ICAM-1 on MDA-MB-231 cells. To identify why MDA-MB-231 cells themselves did not migrate spontaneously through normal human macro- and microvascular endothelial monolayers, we assessed beta -integrin and ICAM-1 expression on MDA-MB-231 cells. Figure 7A shows FACScan analysis of MDA-MB-231 cells stained with control MAbs and different adhesion molecule MAbs, and Fig. 7B illustrates beta 1-, beta 2-, beta 3-integrin and ICAM-1 expression on MDA-MB-231 cells. These tumor cells expressed high levels of ICAM-1 but did not express CD11a/CD18 and CD11b/CD18, two adhesion receptors essential for adhesion to vascular endothelium and migration through the endothelial barrier (6, 19). MDA-MB-231 cells also contained beta 1-integrin, but not beta 3-integrin (Fig. 7, A and B).


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Fig. 7.   A: FACScan analysis of MDA-MB-231 cells stained with FITC- or PE-conjugated negative monoclonal antibodies (MAbs, gray areas) and anti-CD11a, -CD11b, -CD18, -ICAM-1, -beta 1-integrin, and -beta 3-integrin MAbs (black areas) on the basis of a minimum number of 10,000 cells collected. B: illustration of different adhesion receptor expression on MDA-MB-231 cells. The results are presented as log mean channel fluorescence per cell (MCF/cell). ICAM-1, intercellular adhesion molecule-1; data are the means ± SD from 5 separate experiments.

TCM-induced alteration in PMN cytocidal function, receptor expression, and PMN apoptosis. To elucidate the possible mechanisms by which TCM-treated PMN facilitated tumor cell transmigration, we measured PMN adhesion receptor expression and cytocidal function after TCM treatment. Exposure of normal human PMN to TCM for 60 min significantly enhanced CD11b and CD18 receptor expression (P < 0.05 vs. PMN incubated with culture medium) (Fig. 8). Concomitantly TCM significantly suppressed PMN respiratory burst and phagocytosis (P < 0.05 vs. PMN incubated with culture medium) (Fig. 9). These results indicate that TCM-treated human PMN undergo an alteration in phenotype resulting in an increased ability to bind to tumor cells and to migrate through vascular endothelium, but a reduced cytocidal capacity. Furthermore, as demonstrated in Fig. 10, both TCM-treated PMN and migrated PMN had significantly delayed apoptotic rates at 6, 12, and 18 h compared with normal PMN (P < 0.05).


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Fig. 8.   Upregulation of PMN adhesion receptor expression by TCM. Normal human PMN were incubated with TCM or culture medium for 60 min. PMN CD11a, CD11b, and CD18 expressions were assessed as described in MATERIALS AND METHODS and are presented as log MCF/cell. Data are expressed as the means ± SD and are representative of 5 separate experiments. The statistical significance was compared with culture medium-treated PMN, *P < 0.05.



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Fig. 9.   Inhibition of PMN respiratory burst and phagocytosis by TCM. Normal human PMN were incubated with TCM or culture medium for 60 min. PMN respiratory burst and phagocytosis were assessed as described in MATERIALS AND METHODS and are presented as log MCF/cell. Data are the means ± SD from 5 separate experiments. The statistical significance was compared with culture medium-treated PMN, *P < 0.05.



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Fig. 10.   The effect of TCM treatment on PMN apoptosis. Normal human PMN were incubated with TCM or culture medium for 60 min. Migrated PMN were collected at 6 h after transendothelial migration. PMN apoptosis was assessed at different time points by propidium iodide (PI) DNA staining as described in MATERIALS AND METHODS. Data are expressed as the means ± SD from 5 separate experiments. The statistical significance was compared with culture medium-treated PMN, *P < 0.05.

The effect of blocking beta -integrins and ICAM-1 on TCM-treated, PMN-mediated tumor transmigration. Different blocking MAbs were used to identify whether the increased expression of beta 2-integrin (CD11b and CD18) on PMN after TCM treatment and the constitutive expression of beta 1-integrin and ICAM-1 on MDA-MB-231 tumor cells are involved in tumor cell transmigration. CD11b blocking MAb ICRF44, CD18 blocking MAb TS1/18, and ICAM-1 blocking MAb P2A4 significantly attenuated TCM-treated PMN-mediated tumor cell migration (P < 0.05) (Fig. 11). Furthermore, the combination of ICRF44 and P2A4 or TS1/18 and P2A4 resulted in further reduction of tumor cell transmigration. However, beta 1-integrin blocking MAb 4B4 had no effect on TCM-treated, PMN-mediated tumor cell migration.


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Fig. 11.   The effect of different blocking MAbs on TCM-treated PMN- mediated tumor cell migration. Isolated human PMN and MDA-MB-231 cells were pretreated with ICRF44 (10 µg/ml), TS1/18 (10 µg/ml), P2A4 (20 µg/ml), and 4B4 (5.0 µg/ml) for 30 min, and TCM-treated PMN-mediated MDA-MB-231 cell migration after 6 h of incubation was assessed as described in MATERIALS AND METHODS. Data are expressed as the means ± SD from 4 separate experiments. The statistical significance was compared with PMN pretreated with culture medium, *P < 0.05.

The effect of human PMN treated with TCM, LPS, or PMA on tumor cell transmigration, migrated tumor cell viability, and proliferation. To examine the effect of PMN activation on tumor cell transmigration, we stimulated human PMN with TCM; lipopolysaccharide (LPS), which is known to be present in the circulation following surgical insult; or phorbol 12-myristate 13-acetate (PMA, a potent activator of PMN through PKC activation); and compared PMN-assisted tumor cell transmigration and migrated tumor cell viability and proliferation. As shown in Table 1, LPS-activated PMN or PMA-activated PMN also assisted tumor cell transendothelial migration, and the percentages of tumor cell migration were similar to those mediated by TCM-treated PMN. There were no significant differences in migrated tumor cell viability among PMN treated with TCM, LPS, or PMA. However, migrated tumor cells induced by LPS-treated PMN or PMA-treated PMN had a significant reduction in cell proliferation (P < 0.05 vs. TCM-treated PMN).

                              
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Table 1.   Comparison of PMN-assisted tumor cell transmigration, migrated tumor cell viability and proliferation among PMN treated with TCM, LPS, or PMA


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor cell extravasation involves several sequential events that include tumor cell arrest in the microvascular bed of target organs, adhesion to vascular endothelium lining the microvessels, transmigration through the endothelial barrier, attachment to the subendothelial basement membrane, invasion into extracellular matrix by local proteolysis, and finally metastatic colonization in the target organ. Adhesion of tumor cells to vascular endothelium is a prerequisite for tumor cell extravasation. Recent evidence suggests that tumor cell attachment to vascular endothelium is, at least in part, the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium such as granule membrane protein 140 and its ligand (2), connexin43 proteins (5), vascular adhesion molecule-1 (VCAM-1) receptor alpha 4beta 1 and VCAM-1 (16), and selectins (18). Furthermore, several tumor cell lines have been found to impair endothelial integrity by inducing endothelial cell retraction (9) and apoptosis (11) after contact between tumor cells and vascular endothelium, which may facilitate tumor cell extravasation.

Tumor cell transmigration through the endothelial barrier appears to be a critical step in the process of tumor cell extravasation and subsequent metastatic growth. However, the mechanisms involved in this process remain unclear. The human breast adenocarcinoma cell line MDA-MB-231 cell line has been found to be highly metastatic in vivo and highly invasive on reconstituted basement membrane in vitro (3). These tumor cells have also been found to cause endothelial cell apoptosis, thus altering endothelial integrity (11). In the present study, we investigated the hypothesis that these cells may utilize proinflammatory leukocytes to facilitate transmigration. We found that MDA-MB-231 cells failed to cross normal macro- and microvascular endothelial monolayers, suggesting that these tumor cells are unable to complete the process of extravasation independently in this in vitro model. FACScan analysis demonstrated that MDA-MB-231 cells expressed high levels of ICAM-1 and middle levels of beta 1-integrin, but they did not express beta 2-integrins, namely, CD11a/CD18 and CD11b/CD18 and beta 3-integrin. As both CD11a/CD18 and CD11b/CD18 are necessarily required for the process of transendothelial migration (6, 19), the lack of beta 2-integrin expression in MDA-MB-231 cells may, at least in part, account for the failure of these tumor cells to migrate through endothelial monolayers.

The onset and progression of tumor metastasis is a highly complicated and sequential multistep process. Initiation of the metastatic process requires tumor cells to undergo a sequence of extensive interactions with a variety of host cells to achieve metastatic proliferation. Although it is generally agreed that the host-tumor cell interaction is a defensive one, a number of studies have shown that tumor cell-platelet (3, 19) and tumor cell-leukocyte (1, 14) interactions may in fact support tumor cell invasion and the creation of the optimal environment for tumor growth at the metastatic site. Using an in vitro transendothelial model, we have demonstrated in this study that TCM-treated human PMN assisted migration of MDA-MB-231 cells across the endothelial barrier, indicating a supportive role for PMN in tumor cell extravasation and metastasis. Because naive human PMN failed to aid tumor cell transmigration, we wished to investigate the effect of TCM on PMN. We found that TCM suppressed PMN cytocidal function (respiratory burst and phagocytosis) and concomitantly upregulated PMN adhesion receptor (CD11b/CD18) expression. This may indicate that this metastatic breast cancer cell line secretes factors capable of altering the phenotype of PMN to facilitate transmigration while preventing PMN-directed tumor cell damage.

Normal human PMN have a short half-life both in vivo and in vitro consequent to the constitutive activation of an apoptotic cell death program (8). In this study, TCM-treated PMN delayed apoptotic rates. Again, one may speculate that this phenomenon is beneficial to the metastatic process. Although it is uncertain which of the MDA-MB-231 cell-producing soluble factors present in TCM is responsible for altered human PMN function, tumor cell-produced granulocyte-macrophage colony-stimulating factor and/or IL-3 have been found, in previous reports, to be responsible for potentiating tumor metastases induced by tumor-elicited PMN (12).

There are two possible ways that PMN may assist tumor cell migration across an endothelial barrier. The first possibility is that PMN causes endothelial injury by releasing proteases and reactive oxygen species to increase microvascular permeability for tumor cell extravasation, as seen in a tumor-bearing murine model in which PMN-mediated pulmonary vascular injury facilitated tumor cell metastasis in the lung (15). Our results from an endothelial monolayer permeability experiment indicate that the increased MDA-MB-231 cell transmigration mediated by TCM-treated human PMN was not due to the impaired integrity of the endothelial monolayer. The other possibility is an interaction that may occur between PMN and tumor cells in which PMN, through an adhesion receptor-dependent mechanism, may bind to tumor cells and facilitate their migration through vascular endothelium. In the present study, the evidence that MDA-MB-231 cells expressed high levels of ICAM-1 and that TCM upregulated PMN CD11b and CD18 expression indicates that TCM-treated PMN possess the appropriate phenotype to bind and transport tumor cells. The finding that IL-8 increased naive PMN, but not tumor cell migration, suggests that the binding of PMN to tumor cells is a prerequisite for PMN-assisted tumor cell migration. Furthermore, by using different blocking MAbs, we have found that CD11b and CD18 expressed on human PMN and ICAM-1 expressed on MDA-MB-231 cells were involved, at least in part, in PMN-facilitated tumor cell migration. These results support our speculation that TCM-treated PMN-mediated MDA-MB-231 cell transendothelial migration most likely occurs through adhesive interactions between PMN and tumor cells. Furthermore, these cells possess proliferative potential following the migration process, and this finding has potential clinical relevance.

The interesting finding in this study is that activation of human PMN by TCM, LPS, or PMA assists tumor cell transendothelial migration; however, migrated tumor cells mediated by LPS-activated PMN or PMA-activated PMN have an impaired ability to proliferate. Although the exact mechanisms involved are unclear, the possible explanation for this phenomenon may correlate with an increased cytocidal function in PMN after LPS or PMA stimulation. Reactive oxygen species released from LPS-activated PMN or PMA-activated PMN may affect tumor cell proliferation by inhibition of cyclin A.

Together, our data support the concept that the consequence of interactions between PMN and tumor cells may support rather than inhibit tumor progression, since tumor cells can exploit PMN and alter their function to increase their metastatic ability. Confirmation of this finding would have important clinical implications, especially in the perioperative period, when tumor cells are known to be present in the circulation and a transient leukocytosis occurs.


    FOOTNOTES

Address for reprint requests and other correspondence: H. P. Redmond, Dept. of Surgery, Cork Univ. Hospital, Univ. College Cork, Wilton, Cork, Ireland (E-mail: p.redmond{at}ucc.ie).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 March 2000; accepted in final form 9 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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