• cancer;
  • metastasis;
  • lung;
  • rodent;
  • adhesion molecules;
  • e-selectin;
  • neutrophil;
  • sialyl Lewis X


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP-labeled Lewis lung carcinoma subline H-59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide-induced increase in H-59 cell adhesion. Although direct selectin–selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver. © 2009 UICC

Lung cancer is the leading cause of cancer-related deaths worldwide.1 A high proportion of patients harbor metastasis to regional lymph nodes upon presentation, and over half of all patients who undergo curative intent surgical resection of the lung will develop metastatic recurrence.2 The liver is a common site of metastasis for lung cancer, with autopsy studies showing hepatic metastases in over 50% of lung cancer patients.3

The hematogenous dissemination of cancer cells from a primary tumor to distant organs involves a cascade of sequential steps (reviewed in Refs.4,5). The adhesion of circulating tumor cells to the vascular endothelium of distant organs is a key step in the metastatic cascade that may determine the organ tropism and extent of metastasis. Interactions between endothelial selectins and tumor cell selectin ligands have been implicated in the metastatic process.5–8 Selectins are a family of transmembrane glycoproteins possessing an extracellular lectin domain that binds to carbohydrate moieties present on their ligands.9 The three known selectins that function as cell adhesion molecules are expressed on the surface of endothelial cells (E-selectin and P-selectin), platelets (P-selectin) and leukocytes (L-selectin). E-selectin has been shown to contribute to the metastatic spread of multiple cancers, including lung and colon carcinoma, and melanoma. Functional inhibition of E-selectin has been shown to prevent adhesion of tumor cells to endothelial monolayers in vitro, and attenuate the development of metastases in vivo.6, 10, 11 In addition, L-selectin knockout mice were shown to develop fewer lung metastases following intravenous injection of colon carcinoma cells, implying that leukocyte selectin expression, and thus leukocyte–cancer cell interactions, may also play a role in the development of metastasis.12

The ligands recognized by selectins are fucosylated and sialylated carbohydrate antigens such as sialyl Lewis-a (sLea) and sialyl Lewis-x (sLex), which are present on mucin-like glycoproteins.13 sLex and sLea moieties are commonly found on the surface glycoproteins of metastatic cancer cells.14 Numerous groups have reported a correlation between increased surface expression of sLex on tumor cells, a higher rate of metastasis, and a worse prognosis suggesting that these glycoconjugates are functionally relevant to disease progression.14, 15In vitro, down regulation of sLex expression was found to reduce the capacity of tumor cells to adhere to vascular endothelial E-selectin.16

Although the impact of local inflammatory changes in the tumor microenvironment on metastases has been well documented (reviewed in Ref.17), there is emerging evidence to suggest that systemic inflammation may influence the adhesion of circulating tumor cells in the microvasculature of distant organs and thereby metastasis. Investigating the role of systemic inflammation in lung cancer metastasis is highly relevant for several reasons. Surgical resection of the lung remains the cornerstone of any potentially curative treatment plan. However, this operative procedure is associated with surgical trauma resulting in significant systemic inflammation and increased cytokine release.18–20 In addition, high rates of postoperative complications accompany this procedure, with a bacterial pneumonia rate of 10–25% and increased systemic lipopolysaccharide release.21, 22 Several reports from clinical series have raised the possibility that inflammatory consequences resulting from postsurgical complications may influence cancer recurrence.23–25 Evidence from studies of other malignancies such as colorectal carcinoma have shown that proinflammatory cytokines such as TNFα and IL-1β increase tumor cell adhesion to endothelial cells in vitro and in vivo, largely by upregulating the expression of cell adhesion molecules such as selectins on the endothelial surface.26 In addition, soluble inflammatory mediators have been shown to increase the expression of selectin ligands on cancer cells.27–29 Finally, there is emerging evidence, primarily based on in vitro studies, that a third cellular participant, the neutrophil, may contribute to inflammation-facilitated cancer cell adhesion.30–32 Together, these findings suggest that states of systemic inflammation may favor the development of metastases by augmenting the recruitment of circulating tumor cells into organ tissues. However, the role of systemic inflammation in the metastatic spread of lung carcinoma has not been adequately studied in vivo.

Using a murine lung carcinoma model of liver metastasis with a known dependency on selectin–selectin ligand interaction for metastasis, we sought to investigate the contribution of systemic inflammation to the early steps of liver metastasis in vivo. Using intravital microscopy to follow tumor cell arrest during the early stages of liver colonization, we show here that tumor cell recruitment is augmented during states of systemic inflammation and that this effect is mediated, at least in part, by selectin–selectin ligand interactions in neutrophil dependent processes.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References


Six-week-old C57BL/6 mice (Charles River, St. Constant, Quebec) of approximately 25 g were maintained in the Royal Victoria Hospital Animal Facility and used for all the experiments. Mice were anesthetized with a mixture of 200 mg/kg ketamine (Wyeth-Ayerst Canada, Guelph, Ontario) and 10 mg/kg xylazine (Bayer Canada, Etobicoke, Ontario) injected intraperitoneally (ip) and maintained throughout experiments with additional doses administered intravenously (iv) via a jugular vein cannula. All experiments were conducted in accordance with the McGill University Animal Care Committee guidelines.

Cell lines

Lewis lung carcinoma sublines H-59 and M-27, both stably expressing GFP after plasmid transfection, were obtained and cultured as previously described.33 At the time of use, confluent monolayers of H-59 and M-27 cells were detached using a PBS-EDTA solution and resuspended in PBS for injection.

Fluorescence intravital microscopy of the liver (surgery)

Following general anesthesia, the left carotid artery and right jugular vein were each cannulated for tumor cell injection, and administration of additional anesthetic and (where indicated) antibodies/reagents, respectively. The abdomen was opened with a midline incision, and the skin and abdominal wall were removed along the costal margin to the midaxillary line. The falciform ligament was then dissected away from the gallbladder and liver. Animals were placed in a left supine position on a plexiglass stage and the left lobe of the liver was gently positioned on a glass cover slip that was then positioned over the microscope objective. Exposed abdominal tissues were covered in saline soaked gauze to prevent dehydration. The exposed liver lobe was covered with a small square of tissue paper to stabilize the organ and was continuously superfused with warm saline. Animals were maintained at 37°C with an infrared heat lamp throughout the procedure and experiment.

Fluorescence intravital microscopy of the liver (experimental procedure)

The anterior surface of the liver was viewed with an inverted microscope (Nikon TE300, Nikon, Montreal, Quebec) equipped with a 20× objective lens and a video camera (Panasonic Digital KR222, Panasonic, Toronto, Ontario), and images recorded on an iMac G5 (Apple, CA) equipped with video acquisition software. GFP-expressing tumor cells were visualized using epi-fluorescence. Cells that arrested within unoccluded sinusoids of the liver for more than 30 sec were considered adherent, and were counted per microscopic field over six fields of view. In some experiments, leukocyte trafficking was visualized with white-light trans-illumination of the liver using the same intravital microscopy procedure described above. Briefly, one postsinusoidal venule and 7–10 associated sinusoids both above and below the venule were observed. Adherent leukocytes were defined as leukocytes that remained stationary on the venular or sinusoidal endothelium for 30 sec or longer, as previously described.34, 35 Under white-light trans-illumination, leukocytes can be clearly visualized within the vasculature. The numbers of adherent cells within the vessels were counted using video playback analysis of each experiment, and are expressed as the number of adherent leukocytes per 100 μm in postsinusoidal venules or per field of view in sinusoids.

In each experiment, 1.5 × 106 of the indicated cells were injected intra-arterially into the left heart via the carotid artery cannula, and the livers were visualized by fluorescence intravital microscopy 30, 45 and 60 min postinjection. Preliminary experiments (not shown) established that the intra-arterial route of injection provided more consistent measurements as compared with intravenous or intraportal vein injection, and that tumor cell adhesion in the liver was constant between 30 and 60 min following tumor cell administration.

In some experiments, cells were incubated with an anti-sialyl-Lewis x (sLex) mAb (Chemicon, Temecula, CA) or a nonblocking anti-Lewis x antibody (anti-SSEA1; Abcam, Cambridge, MA) as control, before cell injection to block the interaction of these moieties with their endothelial receptors (selectins). In other experiments, mice received 20 mg/kg of the selectin inhibitor fucoidin (Sigma, Oakville, Ontario) 30 min before injection of the tumor cells.

Induction of systemic inflammation

To study the effects of systemic inflammation on tumor cell recruitment, mice received an injection of 0.5 mg/kg lipopolysaccharide (LPS; Sigma, Oakville, Ontario) intravenously via the tail vein 4 hr before cell injection. This concentration of LPS was sufficiently low to allow all animals to survive general anesthesia and perfusion of the liver sinusoids remained such that tumor cell–endothelial interactions occurred under flow conditions. Some animals received 300 μg of RB6-8C5 (anti-Gr1 Ab; Cedarlane, Hornby, Ontario) ip 24 hr before LPS administration, a procedure previously shown to deplete over 97% of circulation neutrophils without affecting other cells in the circulation.36, 37


To investigate the colocalization of GFP expressing H-59 cells and neutrophils, liver specimens were fixed in 4% paraformaldehyde and immersed in 30% sucrose before freezing at −80°C. Specimens were embedded in O.C.T. compound and frozen sections were cut at 10 μm and immunostained with a rat anti-mouse neutrophil specific antibody (L8993AP; Cedarlane, Hornby, ON) followed by Alexa 568-conjugated goat anti-rat (Invitrogen, Burlington, ON). Sections were observed under a laser-scanning confocal microscope (Zeiss, Toronto, ON). The degree of colocalization was measured in LPS-treated mice by counting the percentage of cancer cells with an associated neutrophil per field of view in five random fields for three separate animals.


All data are presented as mean ± SEM. A Mann–Whitney U test was used to determine the significance between population means. Statistical significance was set at p < 0.05.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Intravital microscopy shows that tumor cell arrest during the early stages of metastatic spread correlates with the site specificity of metastasis

H-59 is a Lewis-lung carcinoma subline that is highly metastatic to the liver whereas M-27 is a subline that is spontaneously metastatic to the lung but not to the liver.6 Intravital microscopy was used to quantify tumor cell (H-59 and M-27) adhesion to liver sinusoidal endothelium in vivo, in real time (Fig. 1a). When the number of tumor cells that adhered to the sinusoidal endothelium following injection into the arterial circulation was compared, 14.0 ± 0.37 H-59 cells per field could be observed in the sinusoids, whereas only 1.70 ± 0.31 M-27 cells were seen (Fig. 1b, p < 0.05), suggesting that tumor cell arrest (adhesion) following injection correlated with the metastatic behavior of the cells.

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Figure 1. The adhesion of circulating Lewis lung carcinoma cells within the microcirculation of the liver is a correlate of the metastatic potential. C57BL/6 mice were injected intra-arterially with highly metastatic Lewis-lung carcinoma variant H-59 or nonmetastatic M-27 cells. Fluorescence intravital microscopy was utilized to quantify the number of adherent cells in the liver sinusoids per field of view. (a) A representative field of view is shown with sinusoids and postsinusoidal venules indicated, and adherent GFP-expressing H-59 cells within the sinusoids (scale bar represents 200 μm). (b) Adherent cells were quantified per field of view (×200) within the hepatic sinusoids (M-27, 1.70 ± 0.31; H-59, 14.0 ± 0.37). Data are presented as the arithmetic mean ± SEM of at least five animals injected with H-59 and three with M-27 cells. ***p < 0.05.

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Endothelial cell selectin and tumor cell selectin ligands mediate cell adhesion to the sinusoidal endothelium

Previously, it was reported that mice treated with an anti-E selectin antibody in conjunction with the injection of H-59 cells developed significantly less hepatic metastases, and subsequently tumor cell attachment to hepatic endothelial cell E-selectin was observed using immunohistochemistry and confocal microscopy.6, 33 It was, therefore, of interest to investigate whether selectin ligands containing sialyl-Lewis x (sLex) moieties on the surface of the tumor cells played a role in the early attachment of these tumor cells to sinusoidal endothelial cells. H-59 cells were treated with a monoclonal anti-sLex antibody before injection into the mice. This resulted in a 60% reduction in the number of H-59 cells that adhered within the liver microvessels (Fig. 2), suggesting that sLex-containing glycoproteins were involved in the early attachment of tumor cells to the liver sinusoidal endothelium. Treatment of H-59 cells with an anti-Lewis X (LeX) mAb had no impact on their adhesion in the liver, confirming that the effect of anti-sLeX mAb was specific and attributable to the blockade of sLeX function. Furthermore, when mice were injected intravenously with the selectin-blocking carbohydrate molecule fucoidin before tumor cell inoculation, a similar 60% reduction in the number of adherent tumor cells in the sinusoids was observed (Fig. 2) confirming the involvement of selectins in the early adhesion of tumor cells in the hepatic sinusoids.

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Figure 2. Endothelial selectins and selectin ligands bearing sialyl-LewisX moieties mediate adhesion of circulating Lewis lung carcinoma tumor cells within the liver sinusoids. Liver sinusoids were visualized by fluorescence intravital microscopy to assess the adhesion of H-59 cells in mice untreated (14.0 ± 0.40), or treated intravenously with 20 mg/kg fucoidin (6.2 ± 0.25). Additional untreated mice were inoculated with H-59 cells that were preincubated with an anti-sialyl-Lewisx (sLex) (5.22 ± 0.27) or anti-Lewis X (LeX) mAb (as control; 12.17 ± 0.90) before injection. Data are presented as the means ± SEM of adherent tumor cells within the liver sinusoids per field of view (×200) and based on at least 5 animals per group. ##p < 0.05 relative to the untreated control.

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LPS-mediated systemic inflammation increases the recruitment of circulating tumor cells within the liver sinusoids

Systemic inflammation induced by tissue trauma and/or complications such as bacterial infections following surgical excision of a primary tumor are hypothesized to increase the metastatic potential of residual circulating tumor cells, and as such have been implicated in postsurgical cancer recurrence.24, 38, 39 We hypothesized that the increased metastatic potential observed during systemic inflammation is because of increased recruitment of circulating tumor cells within the microvessels of organs such as the liver. A well described murine model of endotoxemia was therefore employed (intravenous injection of 0.5 mg/kg LPS) to assess the influence of systemic inflammation on tumor cell trafficking within the liver. This model accurately replicates the systemic inflammatory response syndrome seen during severe sepsis, inducing organ dysfunction and significant leukocyte recruitment into the microvessels of the liver and lungs.40

To assess H-59 cell recruitment within the liver sinusoids of systemically inflamed mice, cells were injected into the arterial circulation 4 hr after iv LPS administration, and cell adhesion in the liver was monitored by fluorescence intravital microscopy. We observed a significant increase (>40%) in the adhesion of H-59 cells in the sinusoids of inflamed mice, as compared with untreated controls (Fig. 3).

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Figure 3. Systemic inflammation increases tumor cell adhesion within the liver sinusoids and is attenuated by selectin/sialyl-Lewis X blockade. Liver sinusoids of mice were visualized by fluorescence intravital microscopy to assess the adhesion of H-59 cells in untreated or systemically inflamed (iv LPS) mice per field of view (×200) in five experimental groups. Untreated mice (14.00 ± 0.40) and mice treated intravenously with 0.5 mg/kg LPS (19.7 ± 0.48) were injected with H-59 cells. Pretreatment of tumor cells with neutralizing anti-sialyl Lewis X antibodies (9.71 ± 0.35) or anti-LeX mAb (as control; 20.6 ± 1.21), or pretreatment of mice with fucoidin (20 mg/kg; 9.08 ± 0.28) was employed in some LPS-treated mice. Data are expressed as the means ± SEM of at least five animals per group. ***p < 0.05 relative to untreated controls, ##p < 0.05 relative to LPS-treated controls.

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Selectin–selectin ligand interactions mediate tumor cell adhesion in hepatic sinusoids during systemic inflammation

We next investigated whether the inflammation-induced increase in tumor cell adhesion was also mediated by tumor sLex–host selectin interactions. Pretreatment of H-59 tumor cells with a neutralizing anti-sLex or pretreatment of the mice with fucoidin abolished the LPS-mediated increase in adhesion (Fig. 3) consistent with the results in noninflamed mice (Fig. 2). This suggests that the increase in adhesion because of inflammation was also mediated by a selectin–selectin ligand interaction.

Leukocyte–tumor cell interactions contribute to the increased cancer cell adhesion during systemic inflammation

A hallmark of systemic inflammation is the induction of leukocyte (primarily neutrophil) recruitment into tissues such as the liver and lungs.41 Recruitment of leukocytes from the circulation and subsequent transendothelial migration is induced during inflammation by the upregulation and/or activation of various leukocyte and endothelial cell adhesion molecules. We asked whether sequestration of neutrophils, within the liver microvasculature contributed to the observed increase in tumor cell arrest in systemically inflamed animals, as has been suggested by the in vitro work of others.30, 31 First, the kinetics of LPS-induced leukocyte recruitment within the hepatic vasculature was measured by intravital microscopy as others and we previously described.40, 42, 43 Leukocyte adhesion in sinusoids and postsinusoidal venules increased by 8- and 17.5-fold, respectively, relative to untreated controls (Figs. 4a, 4b). Thus, significant numbers of leukocytes were induced to adhere within the microvessels of the liver in response to the LPS injection. Endothelial E- and P-selectins have been shown to mediate the tethering and rolling of leukocytes within postsinusoidal venules, but are not involved in leukocyte adhesion in the sinusoids.40 Consistent with these previous reports, we observed that an iv injection of fucoidin (selectin blockade) 4 hr after LPS administration prevented leukocyte adhesion in the postsinusoidal venules (Fig. 4a), but did not diminish leukocyte adhesion within the sinusoids of the inflamed liver (Fig. 4b).

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Figure 4. Neutrophil recruitment in the liver is induced during systemic inflammation and attenuated by selectin inhibition. Postsinusoidal venules and seven to ten associated sinusoids above and below each venule were visualized by trans-illumination intravital microscopy to visualize leukocyte trafficking. Shown in (a) are the numbers of adherent leukocytes counted per 100 μm in postsinusoidal venules and in (b) the number of adherent leukocytes in sinusoids per field of view (×400). Each parameter was assessed in the livers of untreated control mice (a, 0.60 ± 0.16; b, 1.30 ± 0.21), LPS-treated control mice (a, 10.53 ± 0.66; b, 10.59 ± 0.64), LPS-treated mice that also received 20 mg/kg intravenous fucoidin (a, 3.57 ± 0.3; b, 11.8 ± 0.6), and mice that received a PMN-depleting antibody (RB6-8C5) 24 hr before LPS injection (a, 0.33 ± 0.21; b, 0.32 ± 0.14). Data are expressed as the means ± SEM of at least five livers analyzed per group. ***p < 0.05 relative to untreated controls, ##p < 0.05 relative to LPS-treated controls.

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Finally, to assess the role of the activated leukocytes in enhancing tumor cell adhesion within sinusoids during inflammation, mice were depleted of circulating polymorphonuclear leukocytes (PMN; neutrophils) before the injection of LPS by the administration of the neutrophil depleting antibody RB6-8C5 24 hr before LPS injection. This treatment is a well-established method to deplete greater than 97% of circulating neutrophils without affecting the numbers of other cell types.36, 44 Previous experiments using rat IgG2b isotype control for RB6-8C5 have shown that neutrophil numbers and function remain unchanged.37, 44 In mice thus treated, leukocyte adhesion in the livers of inflamed mice was abolished (Figs. 4a, 4b). Within the livers of PMN-depleted and LPS-treated mice, the adhesion of circulating H-59 cells was significantly reduced as compared with LPS-treated control mice with normal PMN numbers (Fig. 5a). Therefore, the absence of PMN alone was sufficient to reduce tumor cell recruitment in the livers of systemically inflamed animals. Interestingly, PMN depletion in noninflamed mice also resulted in a small, but statistically significant (p = 0.0041), decrease in H-59 (Fig. 5a). This finding suggests that not only do neutrophils facilitate cancer cell adhesion in both inflamed and noninflamed conditions, but also that cancer cell adhesion in states of systemic inflammation are more dependent on the presence of activated neutrophils.

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Figure 5. Leukocyte-tumor cell interactions contribute to the arrest of circulating tumor cells in the liver sinusoids. (a) Fluorescence intravital microscopy was utilized to quantify the number of adherent H-59 cells per field of view (×200) within the liver sinusoids of untreated control mice (14.0 ± 0.37), LPS-treated control mice (19.68 ± 0.48), control (no LPS) mice that were depleted of circulating PMN by a 24-hr treatment with RB6-8C5 (11.65 ± 0.52; p = 0.0041), LPS-treated mice that were depleted of circulating PMN (8.9 ± 0.40; p < 0.0001), and LPS-treated mice that were depleted of circulating PMN that receive H-59 cells pretreated with sLex blocking mAb (8.63 ± 0.33). Data are presented as the mean ± SEM of at least five livers per group. *p = 0.0037 between PMN depleted mice and LPS-treated PMN depleted mice. ***p < 0.05 relative to untreated mice. ##p < 0.05 relative to LPS-treated mice. (b) Liver tissue sections obtained from LPS-treated mice that were inoculated by the intra-arterial route with 1.5 × 106 GFP-expressing H-59 cells (green) were stained with anti-neutrophil Ab and an Alexa 568 secondary Ab (red) and imaged using confocal microscopy. Images were acquired at ×400 magnification (Left scale bar represents 25 μm, right insert scale bar represents 10 μm).

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The reduction in H-59 adhesion seen with PMN depletion was comparable with that seen with blockade of selectin/sLex interactions (Figs. 2, 5a). We, therefore, sought to determine if selectin/sLex interactions contributed to H-59 adhesion in the absence of PMNs. Combined PMN depletion and sLex blockade did not have an additive effect compared with either treatment alone (Fig. 5a). This finding suggests that neutrophils and selectin-sLex axis binding contribute to the same adhesive process.

To further investigate the link between adherent neutrophils and tumor cell arrest, liver sections from LPS-treated mice that had been injected with GFP-expressing H-59 tumor cells were immunostained with an anti-mouse neutrophil monoclonal antibody and a goat anti-rat Alexa 568 as a secondary antibody and imaged using a confocal microscope. Interestingly, the majority (78 ± 6.6%) of adherent H-59 tumor cells were observed to colocalize with adherent neutrophils, suggesting a contact mediated interaction between these adherent cell types (Fig. 5b).


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Numerous studies have implicated selectin–selectin ligand interactions in the binding of metastatic tumor cells to vascular endothelial cells.6, 10, 45 Moreover, there is mounting evidence that states of systemic inflammation can accelerate the progression of metastatic tumor development.23–25, 38, 39 The present data demonstrate that these two phenomena are inter-related, and that activated neutrophils represent a third cellular participant in this process. In this study, a physiologically relevant in vivo model of the early stages of hematogenous metastasis was developed using fluorescence intravital microscopy to monitor the trafficking of tumor cells in the liver vasculature. We have demonstrated that Lewis lung carcinoma variant H-59 cells adhere readily to liver sinusoids, and that disrupting the interactions between host selectins and tumor cell sLex can prevent tumor cell recruitment to this organ. Furthermore, we provide evidence that systemic inflammation induced by LPS significantly elevated the metastatic potential of lung cancer cells, by increasing the propensity of these cells to arrest within the liver sinusoids. Although selectin–selectin ligand interactions contributed significantly to the sinusoidal adhesion of lung carcinoma cells in the inflamed liver, we show for the first time that activated and adherent neutrophils within the sinusoids also contribute to tumor cell arrest in the liver and the two processes seem to be inter-related.

Previous studies have demonstrated that cultured liver sinusoidal endothelium supports the adhesion of metastatic colorectal carcinoma cells in an E-selectin dependent manner.6 These studies found that the adhesion of tumor cells to a monolayer of liver sinusoidal endothelium, as well as development of hepatic metastases in vivo, could be significantly attenuated by pretreatment of the endothelium with E-selectin blocking antibodies, or by C-raf antisense oligonucleotides (to block the MAPK pathway of E-selectin gene expression).6, 45 Our data suggest that this decrease in liver metastasis induced by selectin inhibition is mediated by attenuating the initial adhesion of H-59 lung carcinoma cells to the sinusoidal endothelium.

E-selectin is normally expressed at low levels on the sinusoidal endothelium, but can be upregulated by factors such as proinflammatory cytokines and bacterial products (LPS).11, 46, 47 Immunohistological examination of tissue samples from patients with metastatic gastrointestinal carcinomas have shown that basal levels of E-selectin expression on liver sinusoidal endothelium is increased during metastatic disease.48 Studies using mouse models of metastasis have shown that the injection of metastatic tumor cells alone is sufficient to rapidly induce production of TNFα, leading to increased expression of E-selectin, as well as other adhesion molecules (VCAM-1, ICAM-1) on the surface of endothelial cells.11, 26 Thus, the presence of malignant tumor cells alone may generate an environment within the circulatory system that is conducive to selectin-mediated cell recruitment within the microvasculature.

In parallel, functional blockade of sLex moieties (selectin ligands) on tumor cells resulted in decreased levels of recruitment into the liver equal to that seen when selectins were inhibited. sLex moieties are commonly found on the surface glycoproteins of many metastatic cancers.14 Numerous groups have reported a correlation between increased expression of sLex on tumor cells and higher rates of metastasis and poor prognosis.49 Clinical studies of colon cancer progression have suggested that the percentage of sLex expressing cells within the primary tumors and the degree of sialylation are prognostic indicators of tumor aggressiveness.14, 15 Furthermore, Zipin et al.16 reported that reducing the surface expression of fucosylated sLex on tumor cells attenuated their capacity to adhere to E-selectin. Our in vivo findings demonstrate that sLex mediated binding contributes to tumor cell recruitment in the sinusoids of the liver, and that targeting these molecules with inhibitory antibodies can potentially reduce metastasis of lung carcinoma cells to this organ.

Assessing the mechanisms of metastasis in a translational context may be highly relevant to lung cancer for a number of reasons. Lung tumors are very aggressive malignancies with many patients presenting with metastatic disease, as well as a high rate of recurrence following curative intent surgical resection.2 Resection of lung cancer often results in significant postoperative inflammation due to the extensive dissection required to remove lung tissue, and a high rate of postoperative infectious complications. Skipper et al.50 showed that tumor recurrence occurs preferentially at sites of trauma, suggesting that wound related factors (hormones, growth factors, cytokines) may promote the growth of micrometastases. In addition, others have suggested that postoperative bacterial infections or inflammation resulting from wound contamination by LPS can enhance metastasis progression.39, 51 Indeed, surgical resection of primary lung cancer frequently involves exposure of sterile tissues to potentially contaminated airways and resulting in transient bacteremia. Previously, it was shown that bacterial LPS can increase colorectal carcinoma adhesion to, and subsequent invasion of cultured endothelial monolayers.52 Thus, we assessed the influence of systemic inflammation on the early events in metastatic progression of lung carcinoma cells in vivo, and found that LPS-mediated systemic inflammation significantly increased the adhesion of H-59 cells within the liver sinusoids. These findings provide in vivo evidence that inflammation can increase the metastatic potential of circulating tumor cells by enhancing their ability to infiltrate the liver.

A number of studies have demonstrated that circulating LPS can activate endothelial cells via Toll-like receptor 4 (TLR4) leading to upregulated expression of adhesion molecules such as E- and P-selectins.45, 52 This suggests that upregulated selectin expression on sinusoidal endothelial cells may account for the augmented adhesion of tumor cells in the liver that we observed during the inflamed state. However, whereas selectin–sLex interactions clearly contributed to tumor cell-endothelial interactions within the liver during systemic inflammation, functional blockade of these molecules did not reduce tumor cell adhesion to the extent that was observed when selectins or their ligands were inhibited in noninflamed mice (Figs. 2 and 3). This implies that upregulation of endothelial selectins was not solely responsible for the augmented adhesive/metastatic potential of circulating tumor cells in systemically inflamed animals. Given this finding, it is likely that other adhesion molecules not identified in this study may be upregulated and/or functionally activated following LPS stimulation to contribute to tumor cell adhesion. The mannose receptor present on the hepatic sinusoidal endothelium is one such molecule that may participate in tumor/endothelial cell interactions, and has been shown to mediate adhesion and liver metastasis with melanoma cells expressing high levels of mannose-type oligosaccharides.53 It is, therefore, possible that the H-59 cell line expresses mannose-type oligosaccharides that may account for the cells remaining after selectin and sLex blockade.

During inflammation, the activation of leukocytes represents a double-edged sword with respect to cancer progression. Although leukocytes possess various antitumor functions, which are currently yielding exciting prospects in the field of cancer vaccines, they have also been shown to support tumor development, progression and metastasis.54 It has been shown in vitro that stationary neutrophils can facilitate tumor cell arrest and transmigration across endothelial monolayers via binding interactions between adherent neutrophils and passing tumor cells that are both adhesion molecule- and shear rate- dependent.30, 31, 55, 56In vivo, we found that systemic inflammation increased tumor cell recruitment in the liver sinusoids when leukocytes simultaneously adhered in these vessels. Indeed, almost 80% of tumor cells were colocalized with adherent neutrophils in the sinusoids of LPS inflamed mice. Although the precise sequence of events has not been definitively determined, these findings suggest that direct binding between adherent neutrophils within liver sinusoids and circulating tumor cells may influence the arrest of tumor cells within these same vessels. Of further interest is the finding that sLex blockade did not result in a step-wise reduction of H-59 adhesion when neutrophils were depleted. This suggests that although selectin–sialyl Lewis X interaction has been shown to independently mediate cancer cell adhesion in vitro,8, 57 in states of systemic inflammation in vivo selectin–sLex mediated cancer cell adhesion and neutrophil facilitated cancer cell adhesion are inter-related.

Neutrophils hypothetically may assist cancer cell adhesion by binding the endothelium resulting in local activation and therefore establishing a fertile ground for cancer cell arrest. Alternatively, neutrophils may also bind cancer cells in circulation directly and form a complex that is more prone to adhesion. Finally, cancer cells may bind the endothelium directly with subsequent arrival of neutrophils. This study is the first to provide an in vivo functional role for neutrophils with respect to cancer cell adhesion within the liver. Indeed, these interactions are likely to be at least in part mediated by sLex interactions with molecules present on neutrophils. Further studies are needed to better characterize the sequence and precise molecular interactions occurring between these cells in vivo.

Taken together, the results presented here provide evidence that adhesion of metastatic lung carcinoma cells in the liver is mediated by interactions between endothelial selectins and tumor cell sLex moieties and that states of systemic inflammation result in unique adhesive mechanisms in the liver sinusoids that rely on both selectin–sLex interactions and activated neutrophils. As blocking of selectin–sLex interactions diminished the adhesion of tumor cells in the liver of noninflamed animals, we propose that these adhesion molecules represent potential therapeutic targets as their inhibition may prevent or attenuate lung cancer metastasis. Identifying the specific molecular players that mediate the interaction between tumor cells and neutrophils during the inflamed state may also yield novel therapeutic targets in the future. Finally, our finding that systemic inflammation increased the metastatic potential of circulating tumor cells by augmenting their adhesion within the liver provides further supporting evidence that anti-inflammatory therapy, particularly in the perioperative period, may be beneficial in the prevention and treatment of cancer and metastatic disease.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
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