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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

A sentinel lymph node (SLN) is the first lymph node that receives drainage from a primary tumor. According to their physiological and biomechanical characteristics, we hypothesized that SLN contains lymphatic endothelial cells (LEC) that are constantly loaded with high levels of shear stress, which might contribute to the production of a suitable environment for micrometastasis within them. To test this hypothesis, we investigated the effects of shear stress stimulation on the expression of adhesion molecules on human LEC isolated from the lymph vessels nearest the SLN of breast cancers, and on the release of ATP from human LEC. The study clarified that the shear stress stimulation produced a significant increase of ICAM-1 expression at protein and mRNA levels in human LEC. Next, we examined whether the shear stress-mediated increase of ICAM-1 expression accelerates the attachment of carcinoma cells to human LEC. Finally, in in vivo experiments, we evaluated whether exogenous ATP facilitates the expression of carcinoma cell-ligated adhesion molecules in rat SLN. In conclusion, shear stress stimulation induces ICAM-1 expression on human LEC by activating cell surface F1/FO ATP synthase, which might contribute to the development of a premetastatic environment within SLN. (Cancer Sci 2012; 103: 1245–1252)

A sentinel lymph node (SLN) is the first lymph node that receives drainage from a primary tumor. Morton et al.[1] initially demonstrated the SLN concept in a feline model and later confirmed it in a clinical study of patients with breast cancer and melanoma. The SLN concept has subsequently become one of the most important topics in surgical oncology.[2, 3] SLN is also the most common and earliest site of malignant tumor metastasis.

Many factors, such as the humoral, immunological and morphological heterogeneity of collecting lymph vessels, might contribute to deciding which lymph node within the regional lymph network draining a primary tumor becomes the SLN. However, it remains unclear which factors play pivotal roles in deciding which lymph node becomes the SLN. In addition, we consider SLN to be constantly subjected to a high lymph flow rate. We reached this conclusion in our recent study, in which a high lymph flow rate was detected within the SLN of the stomach using contrast-enhanced ultrasonography with Sonazoid (Daiichi-Sankyo Group, Tokyo, Japan) in a porcine model.[4] Thus, the endothelial cells in the marginal sinuses of SLN and its nearest afferent lymph vessels might be constantly stimulated by the high shear stress generated by the abovementioned high lymph flow rate because collapsible tubes, such as the collecting lymph vessels, display unchanged diameters in such conditions. Therefore, we have hypothesized that SLN contains lymphatic endothelial cells (LEC) that are constantly loaded with a high level of shear stress.

Vascular endothelial cells (EC) are constantly exposed to shear stress, the mechanical force generated by the flow of blood or lymph. Upon detecting shear stress, these EC transmit signals to their interiors, where they trigger cell responses, including changes in a variety of cell functions.[5, 6] In the case of LEC, lymph flow stimulation results in the rapid production and release of endogenous nitric oxide (NO), which contributes to the cyclic GMP-mediated relaxation of lymphatic smooth muscles[7] and inhibitory effects on lymph pump activity.[8] We have also demonstrated that in response to shear stress, LEC release endogenous ATP, which activates endothelial constitutive NO synthase (ecNOS) expression at the mRNA and protein levels through the inositol 1,4,5-triphosphate-mediated release of intracellular Ca2+ and the activation of Ca2+-activated K+ channels in LEC.[9]

Kaplan et al. and Hiratsuka et al.[10, 11] show that primary tumors influence the microenvironments of distant organs during the development of metastasis. However, it is unclear whether similar environmental changes occur within SLN to encourage carcinoma micrometastases to develop within them. If such environmental changes occur within SLN, the mechanisms responsible for these changes need to be elucidated. Therefore, to address the pathophysiological roles of shear stress stimulation in the development of environmental changes within the SLN, we attempted to investigate: the effects of shear stress on the expression of adhesion molecules on cultured human LEC isolated from the afferent lymph vessels nearest to SLN and on the release of ATP from human LEC; and to study whether shear stress-mediated increases in adhesion molecule expression accelerate the attachment of carcinoma cells to cultured human LEC. Finally, in in vivo rat experiments, we evaluated whether the ATP released from LEC in response to shear stress stimulation facilitates the expression of carcinoma cell-ligated adhesion molecules within rat SLN.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell culture

The isolation and culturing of the human LEC were performed according to the techniques described by Kawai et al.[9] using the afferent lymph vessels nearest to the sentinel lymph nodes in breast cancer patients. The human LEC experiments were approved by the ethical committee for human studies of Shinshu University School of Medicine. All subjects were informed of the risks and purpose of the study before their written consent was obtained. The human LEC were maintained in endothelial growth medium (EGM)-2 supplemented with 10% FBS and used at the fourth to ninth passage.

The human breast adenocarcinoma cell line MDA-MB-231 was purchased from the American Type Culture Collection (Manassas, VA, USA). The carcinoma cells were maintained in DMEM/Nutrient Mixture F12 Ham (DMEM/F12) culture medium supplemented with 1% FBS. The human LEC were incubated under atmospheric conditions of 5% O2, 5% CO2 and 90% N2 at 37°C, whereas the carcinoma cells were incubated under normoxic conditions of 21% O2, 5% CO2 and 74% N2 at 37°C.

Flow loading experiments

A parallel plate-type apparatus was used to apply laminar shear stress to the human LEC using a slightly modified version of Yamamoto's method.[6] Briefly, one side of the flow chamber consisted of a 1% gelatin-coated glass plate, on which the cultured human LEC rested, and the other side consisted of a polycarbonate plate. These two surfaces were held 200 μm apart with a Teflon gasket. The chamber contained an entrance and an exit for the fluid, and the entrance was connected to an upper reservoir by a silicone tube. The exit led to a lower reservoir. The flow of the fluid (37°C, EGM-2) was driven by a peristaltic pump with a flow rate controller. The intensity of the shear stress (τ, dynes/cm2) acting on the LEC layer was calculated using the formula τ = 6μQ/a 2 b, where μ is the viscosity of the perfusate (poise), Q is the flow volume (mL/s), and a and b are the cross-sectional dimensions of the flow path (cm).

ATP assay

The ATP concentrations of the human LEC culture media supernatants were determined using a luciferin–luciferase assay based on the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA), as described previously.[12] Briefly, to develop a calibration curve for ATP measurement, we first established a luciferin–luciferase assay using culture media containing several concentrations of ATP. Next, 100 μL of the flow-stimulated human LEC supernatant was collected in a 96-well plate, into which 100 μL of luciferin–luciferase solution had been added, and light emission was recorded using a luminometer (Dainippon Sumitomo Pharma, Osaka, Japan). Thus, we calculated the concentrations of ATP in the supernatants of the flow-stimulated human LEC culture media using the calibration curve for ATP measurement.

Immunocytochemistry

Indirect immunocytochemical studies were performed using cultured LEC seeded on glass slides coated with type I collagen, and then the cells were fixed with 4% paraformaldehyde in PBS solution for 20 min at room temperature. Next, the cells were washed three times with PBS and then incubated overnight at 4°C with primary polyclonal human antisera to E-selectin/CD62E (dilution: 10 μg/mL; R&D Systems, Minneapolis, MN, USA), P-selectin/CD62P (dilution: 10 μg/mL; R&D Systems), VCAM-1/CD106 (dilution: 10 μg/mL; R&D Systems), ICAM-1/CD54 (dilution: 10 μg/mL; R&D Systems) and ATP synthase (dilution 1:50; Millipore, Billerica, MA, USA). After being washed three times in PBS, the cells were incubated for 1 h at room temperature with 1:100 diluted Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA). The nuclei of the cultured cells were counterstained and mounted with ProLong Gold antifade reagent and DAPI (Molecular Probes, Eugene, OR, USA), before being examined with a fluorescent microscope (Leica, Wetzler, Germany) and photographed.

To quantitatively evaluate the data obtained in the immunocytochemical studies, in each experiment we examined the cultured cells in more than three culture dishes and then took three photographs of each culture dish. Each image contained 7–11 cells; therefore, approximately 100 cultured cells were examined.

For non-specific staining, the primary antiserum was replaced with Block-ace (Dainippon Sumitomo Pharma) as a negative control.

Quantitative RT-PCR

The expression of ICAM-1 mRNA was evaluated using quantitative RT-PCR. Total RNA was extracted from the cultured human LEC using the ISOGEN reagent (Nippon Gene, Toyama, Japan), according to the manufacturer's instructions. The concentration of each RNA was calculated by measuring the absorbance of the RNA samples at 260 nm with a spectrophotometer. The extracted RNA was reverse-transcribed with M-MLV reverse transcriptase (Ambion, Austin, TX, USA). For RT-PCR analysis, each superscript first-strand synthesis kit (Invitrogen) was used with 1.0 μg of total RNA. The following forward and reverse primers for ICAM-1 and cyclophilin A were used for each specific probe: ICAM-1 (Takara, Kyoto, Japan) and cyclophilin A: 5′-TTCGTGCTCTGAGCACTGGAG-3′ (forward) and 5′-GGACCCGTATGCTTTAGGATGAAG-3′ (reverse). The cDNA was diluted fivefold prior to PCR amplification. Quantitative RT-PCR was performed using a Light Cycler rapid thermal cycler system (Roche Diagnostics, Burgess Hill, UK). The reactions were performed in a 20-μL volume with 0.5 μM primer, Taq DNA polymerase and the buffer included in the SYBR Premix Ex Taq kit (Takara). The PCR protocol included a 10-s denaturation step followed by 45 cycles of denaturation for 5 s at 9°C and annealing for 20 s at 60°C. The fluorescent product was detected at the end of the 60°C extension period. Negative controls, including the PCR reaction products each primer pair produced from, were subjected to melting curve analysis. The data were analyzed using the Light Cycler analysis software. The results are presented as ratios of the expression of ICAM-1 mRNA to that of cyclophilin A.

In vitro human LEC attachment assay

Human LEC were plated on type I collagen-coated 35-mm plates and then incubated in 5% O2, 5% CO2 and 90% N2 at 37°C to form confluent monolayers. The LEC were kept in serum-starved EBM-2 medium supplemented with 3% FBS. Selected plates were treated with 10−7 M ATP or shear stress stimulation (1.0 dyn/cm2) for 2 h. In some experiments, 10−6 M suramin was simultaneously added to the plates during 2 h treatment with 10−7 M ATP or shear stress stimulation.

In some experiments, the plates were also treated with 10 μg/mL anti-human ICAM-1 antibody (R&D Systems) for 30 min after 2 h treatment with the shear stress stimulation or 10−7 M ATP. Breast cancer cells that had been stained with PKH26 fluorescent dye (Sigma, St Louis, MO, USA) were then plated at 5 × 104 cells per plate and incubated for 30 min at 37°C. Unbound cells were removed by aspiration, and the plates were washed three times with DMEM/F12. Attachment was quantitated by counting the number of attached cells under ×100 magnification using a Leica microscope.

In vivo immunocytochemical experiments

Male Sprague–Dawley rats (6–8 weeks old; n = 4; body weight 142 ± 5 g; Japan SLC, Hamamatsu, Japan) were used for the present study. The rats were housed in an environmentally-controlled vivarium and fed a standard pellet diet and water ad libitum. All experimental protocols were approved by the animal ethics committee of Shinshu University School of Medicine in accordance with the Japanese Physiological Society's “Guide for the Care and Use of Laboratory Animals.” As in vivo model experiments used to evaluate the effects of shear stress on LEC within SLN, the rats received 1.0 mL PBS (negative control) or 1.0 mL PBS containing 10−6 M ATP in the roots of their tails. At 24 h after the administration of the test chemicals, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.; Tokyo Chemical Industry, Tokyo, Japan) and exsanguinated. Their iliac lymph nodes were rapidly isolated and fixed with liquid nitrogen for the immunocytochemical studies. Slices of the tissue samples were then fixed with 100% acetone and permeabilized with 0.1% Triton X-100. Next, the slices were washed three times with PBS and incubated for 2 h at room temperature with primary polyclonal rat antiserum to 1:20 diluted FITC-labeled ICAM-1/CD54 (R&D Systems). After being washed three times in PBS, the tissue slices were mounted with ProLong Gold antifade reagent and DAPI (Molecular Probes) to counterstain the cell nuclei in the lymph nodes, examined with a fluorescent microscope (Leica), and photographed.

In vivo intravital experiments

Male Sprague–Dawley rats (6–8 weeks old; n = 4; body weight 140 ± 6 g; Japan SLC) were used for these experiments. The rats were housed in an environmentally-controlled vivarium and fed a standard pellet diet and water ad libitum. All experimental protocols were approved by the animal ethics committee of Shinshu University School of Medicine in accordance with the Japanese Physiological Society's “Guide for the Care and Use of Laboratory Animals.” In the in vivo model experiments, the rats were also given 1.0 mL PBS subcutaneously (negative control) or 1.0 mL PBS containing 10−6 M ATP. At 24 h after the administration of the test chemicals, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.; Tokyo Chemical Industry). Additional anesthetic was added intramuscularly as needed. A tracheostomy was performed to ensure that the airway remained patent. The right carotid artery was cannulated to allow arterial blood pressure monitoring. The left external carotid vein was also cannulated to allow the continuous intravenous administration of isotonic saline solution (30 μL/min/100 g body weight) throughout the experiments.

After an incision had been made in the abdomen, the iliac lymph nodes and the afferent and efferent lymph vessels were isolated from the surrounding adipose and connective tissue and then placed in a custom-made chamber for intravital video microscopic observation (Leica). The equipment did not obstruct the flow of lymph through the vessels. The fluorescent changes within the lymph nodes were evaluated time-dependently after the subcutaneously administration of 0.3 mL FITC-labeled ICAM-1 antibody diluted 1:10 with PBS containing 1% BSA into the roots of the rats' tails.

Drugs

All salts were obtained from Wako (Tokyo, Japan), and ATP, suramin, piceatannol and acetazolamide were purchased from Sigma. The piceatannol was diluted in ethanol. The concentration of ethanol used did not affect the biological viability of the cultured EC. Drug concentrations are expressed as the final concentration in the culture plate.

Statistical analyses

All results are expressed as the mean ± SEM. Statistical significance was analyzed with the Student's t-test for unpaired observations or one-way anova, followed by Duncan's post-hoc test, as appropriate. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Effects of shear stress stimulation strength and duration on the expression of adhesion molecules on human lymphatic endothelial cells

First, to evaluate the antibody specificity controls used for the immunocytochemical analyses, we stained the adhesion molecules (E-selectin, P-selectin, VCAM-1 and ICAM-1) on cultured human LEC stimulated by 2 h treatment with 10 ng/mL LPS (positive control). Significantly high expression of E-selectin and ICAM-1, but only weak expression of P-selectin and VCAM-1, was detected on the LEC (Fig. 1A,m–p). In preliminary experiments, we found that stimulation with a shear stress of 1.0 dyn/cm2 significantly increased the ICAM-1 immunocytochemical and mRNA expression levels of human LEC. In the present experiments, the expression of adhesion molecules on human LEC was measured at different time points up to 24 h after their stimulation with a shear stress of 1.0 dyn/cm2. Figure 1(A) shows representative photomicrographs of the effects of shear stress stimulation for different time periods on the expression of adhesion molecules on human LEC. At 0 h, little or no expression of E-selectin, P-selectin, VCAM-1 or ICAM-1 was observed on the cultured human LEC (Fig. 1A,a–d). The photomicrographs were merged with nuclear staining images of the same cultured cells. Overnight culturing of the cells in starvation medium containing 3% FBS induced little or no adhesion molecule expression on human LEC (negative control). In contrast, 2 or 24 h stimulation with a shear stress of 1.0 dyn/cm2 selectively increased the expression of ICAM-1 on human LEC (Fig. 1A,h,l). However, little or no expression of E-selectin, P-selectin or VCAM-1 was observed on the human LEC subjected to 2 or 24 h stimulation with a shear stress of 1.0 dyn/cm2 (Fig. 1A,e–g,i–k). The ICAM-1 expression results are shown in Figure 1(B). Thus, stimulation with a shear stress of 1.0 dyn/cm2 for 2 h or more significantly increased the expression of ICAM-1 on human LEC.

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Figure 1. (A) Representative photomicrographs of the effects of starvation culture medium containing 3% FBS during 24 h culture (a–d), shear stress stimulation (1.0 dyn/cm2) for different lengths of time (2 or 24 h) (e–l), or 2 h treatment with 10 ng/mL LPS (m–p) on the expression of adhesion molecules; that is, E-selectin, P-selectin, VCAM-1 and ICAM-1, on cultured human lymphatic endothelial cells (LEC). The photomicrographs have been merged with nuclear staining images of the same cells. Each marker represents 50 μm. (B) The density measurements of each photomicrograph, which were obtained using Scion Image analysis. The ordinate denotes the normalized value of each density measurement shown as the mean density/pixel (n = 5). The abscissa shows the length of the shear stress stimulation period. **P < 0.01, significantly different from the no shear stress stimulation-mediated response. (C) The effects of shear stress strength (0.5 or 1.0 dyn/cm2) on ICAM-1 expression on human LEC. The ordinate represents the same item as in (B). The abscissa shows the strength of the shear stress stimulus. **P < 0.01, significantly different from the no shear stress stimulation-mediated response. (D) The effects of shear stress strength on ICAM-1 mRNA expression in human LEC, as evaluated by RT-PCR. The ordinate shows the ratio of ICAM-1 signals to cyclophilin A signals (n = 4). The abscissa shows the strength of the shear stress stimulus. **P < 0.01, significantly different from the no shear stress stimulation-mediated response.

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Figure 1(C) indicates the effects of shear stress strength on the expression of ICAM-1 on human LEC. Two hours stimulation with a shear stress of 0.5 or 1.0 dyn/cm2 significantly increased the expression of ICAM-1 on human LEC in a strength-dependent manner.

In addition, the ICAM-1 mRNA expression in the human LEC was also significantly increased in response to 2 h stimulation with a shear stress of 0.5 or 1.0 dyn/cm2 (Fig. 1D).

Effects of piceatannol or suramin on the shear stress stimulation-mediated increase of ICAM-1 expression

To address the molecular mechanisms responsible for the shear stress stimulation-mediated increases of ICAM-1 expression at protein and mRNA levels, we investigated the effects of the cell surface F1/FO ATP synthase inhibitor piceatannol[6, 13] and the non-selective purinergic P2X/P2Y inhibitor suramin[12] on the shear stress stimulation (1.0 dyn/cm2, 2 h)-mediated increases of ICAM-1 expression in human LEC. Figure 2(A,1–3) shows representative photomicrographs of shear stress stimulation-induced ICAM-1 expression on human LEC in the absence or presence of 20 μM piceatannol or 10−6 M suramin. Two hours stimulation with a shear stress of 1.0 dyn/cm2 significantly increased the expression of ICAM-1 on human LEC (Fig. 2A,1). Pretreatment with 20 μM piceatannol or 10−6 M suramin produced a significant reduction in the shear stress stimulation-mediated increase of ICAM-1 expression on human LEC (Fig. 2A,2,3). These results are shown in Figure 2(B,1–3). Pretreatment with piceatannol or suramin also caused a significant reduction in the shear stress-mediated increase in ICAM-1 mRNA expression in human LEC (Fig. 2C,2,3). These results are demonstrated in Figure 2(C,1–3).

image

Figure 2. (A) 1–3, Representative photomicrographs of the effects of shear stress stimulation (1.0 dyn/cm2, 2 h) on ICAM-1 expression on human lymphatic endothelial cells (LEC) in the absence or presence of 20 μM piceatannol (a selective inhibitor of cell surface F 1/F O ATP synthase) or 10−6 M suramin (a non-selective inhibitor of purinergic P2X/P2Y receptors). (A) 4–5, Representative photomicrographs of the effects of exogenous ATP (10−6 M) on ICAM-1 expression on human LEC in the absence or presence of 10−6 M suramin. The photomicrographs have been merged with nuclear staining images of the same cultured cells. Each marker represents 50 μm. (B) The density measurements of each photomicrograph, which were obtained using Scion Image analysis. The ordinate represents the same item as in (B). Each number on the abscissa corresponds to the item with the same number in (A). *P < 0.05, significantly different from the shear stress stimulation (1.0 dyn/cm2, 2 h)-mediated (positive control) response (column 1). **P < 0.01, significantly different from the exogenous ATP (10−6 M, positive control)-mediated response (column 4). (C) The effects of shear stress stimulation (1.0 dyn/cm2, 2 h) in the absence or presence of 20 μM piceatannol or 10−6 M suramin, and exogenous ATP (10−6 M) in the absence or presence of 10−6 M suramin, on ICAM-1 mRNA expression in human LEC, as evaluated by RT-PCR. Each number on the abscissa represents the same item as in (A). The ordinate represents the same item as in Figure 1(D). *P < 0.05, significantly different from the shear stress stimulation (1.0 dyn/cm2, 2 h)-mediated response (column 1). **P < 0.01, significantly different from the exogenous ATP (10−6 M)-mediated response (column 4).

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Exogenous ATP induces ICAM-1 expression at the protein and mRNA levels in human lymphatic endothelial cells

To evaluate the hypothesis that shear stress stimulation induces ATP secretion from human LEC by activating F1/FO ATP synthase, which might contribute to the shear stress stimulation-mediated increases in ICAM-1 protein and mRNA expression in human LEC, we first examined the effects of exogenous ATP on the expression of ICAM-1 in human LEC in the absence or presence of suramin. As shown in Figure 2(A,4), 2 h treatment with 10−6 M exogenous ATP produced a significant increase in ICAM-1 expression on human LEC. The increase was similar to that produced by shear stress stimulation (1.0 dyn/cm2, 2 h) (Fig. 2A,1). The exogenous ATP-mediated increase of ICAM-1 expression on human LEC was significantly reduced by simultaneous treatment with 10−6 M suramin (Fig. 2A,5). These results are shown in Figure 2(B,4,5).

The exogenous ATP also produced a significant increase in ICAM-1 mRNA expression in human LEC. The ATP-induced increase in ICAM-1 mRNA expression was significantly reduced by treatment with 10−6 M suramin. These results are shown in Figure 2(C).

Effects of piceatannol on shear stress stimulation-mediated ATP release from human lymphatic endothelial cells

We next measured the concentrations of ATP in the culture supernatants of shear stress stimulation-loaded human LEC. Figure 3(A) shows the results of these experiments. Two hours stimulation with a shear stress of 0.5 or 1.0 dyn/cm2 produced a significant increase in the concentration of ATP. The shear stress stimulation-induced release of ATP from the human LEC was significantly reduced by pretreatment with 20 μM piceatannol (Fig. 3A).

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Figure 3. (A) The effects of shear stress stimulation (0.5 or 1.0 dyn/cm2, 2 h) on the concentration of ATP in the human lymphatic endothelial cells (LEC) culture supernatant in the absence or presence of 20 μM piceatannol. The ordinate indicates the concentration of ATP in the culture supernatant, which was measured in a luciferin–luciferase assay. The abscissa denotes shear stress strength. *P < 0.05, **P < 0.01, significantly different from the no shear stress stimulation-mediated response (negative control) or the 1.0 dyn/cm2 shear stress stimulation-mediated response (positive control). (B) Representative photomicrographs of the effects of shear stress stimulation (1.0 dyn/cm2, 2 h) on the cell surface F 1/F O ATP synthase expression of non-permeabilized cultured human LEC. The photomicrographs have been merged with nuclear staining images of the same cells. Each marker represents 50 μm. (C) The density measurements of the photomicrographs shown in Figure 2(E), which were obtained using Scion Image analysis. The ordinate denotes the normalized value of each density measurement shown as the mean density/pixel (n = 5). NS, no significant difference between the columns.

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To confirm the existence of cell surface F1/FO ATP synthase on human LEC, we next immunocytochemically investigated the expression of F1/FO ATP synthase on non-permeabilized cultured cells. Figure 3(B) shows representative photomicrographs of cell surface F1/FO ATP synthase molecules on the surfaces of non-permeabilized human LEC that had been loaded without or with shear stress stimulation (1.0 dyn/cm2, 2 h). We confirmed that F1/FO ATP synthase was present on the surfaces of human LEC. However, shear stress stimulation had little or no effect on F1/FO ATP synthase protein expression on the surfaces of human LEC. Figure 3(C) shows these results.

Attachment assay involving 2 h stimulation with shear stress or exogenous ATP in the absence or presence of suramin

As shown in Figure 4(A,1) (negative control) and Figure 4(A,2), 2 h shear stress stimulation (1.0 dyn/cm2) caused a significant increase in the attachment of carcinoma cells to human LEC. The increased attachment of carcinoma cells to human LEC was significantly reduced by simultaneous treatment with 10−6 M suramin (Fig. 4A,3). These results are shown in Figure 4(B,1–3).

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Figure 4. (A) Representative dark-field photomicrographs of the in vitro attachment assay assessing the effects of 2 h shear stress stimulation (A2; 1.0 dyn/cm2, positive control) or 2 h treatment with exogenous ATP (A5; 10−6 M, positive control) in the absence or presence of 10−6 M suramin (A3 and 6, respectively) or anti-ICAM-1 antibody (A4 and 7, respectively). (B) Attachment assay data, which were normalized to the number of adherent carcinoma cells per field (×100). **P < 0.01, significantly different from data obtained during shear stress stimulation (A2 and B2; positive control) or after the addition of exogenous ATP (A5 and B5; positive control). **P < 0.01, significantly different from the negative control (A1 and B1; stimulation with starvation culture medium alone).

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Similar to shear stress stimulation, 2 h stimulation with 10−6 M exogenous ATP produced a significant facilitation of the attachment of carcinoma cells to human LEC (Fig. 4A,5). In addition, simultaneous treatment with 10−6 M suramin caused a significant reduction in the exogenous ATP-mediated increase in the attachment of carcinoma cells to human LEC (Fig. 4A,6). These results are shown in Figure 4(B,5,6).

Attachment assay involving 2 h stimulation with shear stress or exogenous ATP in the absence or presence of anti-ICAM-1 antibody

Next, we examined whether shear stress stimulation-mediated or exogenous ATP-mediated facilitation of the attachment of carcinoma cells to human LEC could be blocked by treatment with 10 μg/mL anti-ICAM-1 antibody. As shown in Figure 4(A,2,4), 2 h shear stress (1.0 dyn/cm2) stimulation produced a significant increase in the attachment of carcinoma cells to human LEC, which was also significantly reduced by treatment with anti-ICAM-1 antibody. These experimental results are shown in Figure 4(B,2,4). Similar to the effects of shear stress stimulation, 2 h stimulation with 10−6 M exogenous ATP also caused a significant increase in the attachment of carcinoma cells to human LEC (Fig. 4A,5,7). The increased attachment of carcinoma cells to human LEC was significantly reduced by additional treatment with anti-ICAM-1 antibody (Fig. 4B,5,7).

ATP-mediated increase of ICAM-1 expression in in vivo rat iliac lymph nodes

To evaluate whether shear stress stimulation could produce ICAM-1 expression in in vivo lymph nodes by inducing the release of ATP via the activation of cell surface F1/FO ATP synthase on LEC, we investigated the effects of subcutaneously-administered exogenous ATP (10−6 M) on ICAM-1 expression within the regional lymph nodes (around SLN) in in vivo experiments. As shown in Figure 5(A), the subcutaneous administration of 1.0 mL 10−6 M exogenous ATP into the rats' tail roots at 24 h before starting the experiments produced a marked increase in ICAM-1 expression in the rats' iliac lymph nodes. However, the administration of 1.0 mL PBS (negative control) induced little or no ICAM-1 expression in these lymph nodes (Fig. 5A, left panel). These results are shown in Figure 5(B) (n = 10 images).

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Figure 5. (A) Representative photomicrographs of the effects of the subcutaneous administration of 1.0 mL of exogenous ATP (10−6 M; right panel) or PBS (left panel) on ICAM-1 expression within rat iliac lymph nodes in in vivo experiments. The lymph nodes were isolated and fixed with liquid nitrogen at 24 h after the administration of ATP. The photomicrographs have been merged with nuclear staining images of the same cells. Each marker represents 200 μm. (B) Density measurements of the photomicrographs shown in (A), which were obtained using Scion Image analysis. The ordinate represents the same item as in Figure 3(C) (n = 10 images). **P < 0.01, the columns are significantly different. (C) Representative photographs the effects of the subcutaneous administration (1.0 mL) of FITC-labeled anti-ICAM-1 antibody on the appearance of ICAM-1-ligated fluorescence within rat iliac lymph nodes in in vivo experiments. The lymph nodes had been perfused with 1.0 mL PBS containing no (negative control; left panel) or 10−6 M exogenous ATP for 24 h before the intravital microscopic observation. The upper and lower panels show optical and fluorescent photomicrographs, respectively. Each marker represents 1 mm. The black and white arrows indicate murine iliac lymph vessels. (D) The density measurements of the photomicrographs shown in (C), which were obtained using Scion Image analysis. The ordinate represents the same item as in Figure 3(C) (n = 10 images). **P < 0.01, the columns are significantly different.

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Similar to the in vivo rat SLN immunocytochemical experiments, the subcutaneous administration of 1.0 mL FITC-labeled anti-ICAM-1 antibody into the roots of the rats' tails demonstrated that the iliac lymph nodes, which had been perfused with 1.0 mL PBS containing 10−6 M exogenous ATP through their afferent lymph vessels for 24 h, displayed significantly increased ICAM-1 expression (Fig. 5C, right panel) around 10 min after the administration. However, in the rat lymph nodes that were perfused with PBS alone (negative control) for 24 h, little or no anti-ICAM-1 antibody fluorescence was observed within the iliac lymph nodes (Fig. 5C, left panel). These results are shown in Figure 5(D) (n = 10 images).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Shear stress stimulation contributes to the production of a suitable environment for micrometastasis within sentinel lymph nodes

Sentinel lymph nodes (SLN) are the most common and earliest site of malignant tumor metastasis. The clinical success of sentinel node navigation surgery suggests that SLN are an effective mechanical barrier against migrating cancer cells.[14] SLN also contain marginal endothelial cells, which might be constantly loaded with high shear stress. It is known that primary tumors influence the microenvironments of distant organs during the development of metastasis.[10, 11] However, it is unclear which molecules in premetastatic SLN loaded with high shear stress produce a suitable environment for micrometastasis within the node. Thus, we examined the hypothesis that the high shear stress generated by increased lymph flow through the SLN and its afferent lymph vessels contributes to the development of a premetastatic environment that is suitable for carcinoma micrometastases within the node.

The major findings of this study are summarized as follows. Two hours stimulation of cultured human LEC with a shear stress of 0.5 or 1.0 dyn/cm2 caused a significant increase in ICAM-1 expression in human LEC. The shear stress stimulation strength used in the present study has been physiologically detected in vivo on the LEC of collecting lymph vessels.[7] Stimulation with the same level of shear stress resulted in the release of a significant amount of ATP through the activation of cell surface F1/FO ATP synthase on human LEC. In agreement with these findings, F1/FO ATP synthase was immunocytochemically confirmed to be present on the surfaces of human LEC. In addition, the administration of the selective cell surface F1/FO ATP synthase inhibitor piceatannol (20 μM) caused a significant reduction in the shear stress stimulation-induced increases of the ICAM-1 expression at protein and mRNA levels in human LEC. These findings were caused by a piceatannol-mediated reduction in shear stress stimulation-induced ATP release from the cells. In addition, shear stress stimulation caused a significant increase in the attachment of carcinoma cells to human LEC. The exogenous addition of ATP also produced a similar response in the carcinoma cell and human LEC attachment assay. Pretreatment with anti-ICAM-1 antibody or the non-selective purinergic P2X/P2Y inhibitor suramin caused a significant reduction in the shear stress stimulation-mediated and exogenous ATP-mediated increases in the attachment of carcinoma cells to human LEC. In agreement with the findings obtained in the present molecular biological studies of human LEC, the expression of ICAM-1 within rat SLN that had been pretreated with exogenous ATP was confirmed both immunocytochemically and using intravital fluorescent microscopy. In conclusion, shear stress stimulation generated by the high rate of lymph flow through SLN and their afferent lymph vessels produced a significant increase in ICAM-1 expression on the surfaces of LEC via the activation of cell surface F1/FO ATP synthase, which might contribute, in part, to the creation of a premetastatic environment and the subsequent production of carcinoma micrometastases within SLN. This conclusion is shown schematically in Figure 6. To confirm this conclusion, it will be necessary to evaluate the effects of shear stress stimulation on ICAM-1 expression within the SLN in an in vivo animal model study.

image

Figure 6. Schematic diagram outlining the conclusion of the present study.

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Crucial roles of ICAM-1 in micrometastasis

ICAM-1 expression by tumor cells is a major contributor to the facilitation of metastatic progression.[12, 15] Recently, we also observed strong ICAM-1 expression in human breast cancer SLN tissue that had been subjected to carcinoma cell micrometastasis, but weak or no ICAM-1 expression in SLN tissue that had not been subjected to metastasis.[16] In contrast, studies of leukocyte-endothelial cell adhesion in tumor microvessels have demonstrated diminished adhesive interactions under both basal and cytokine-stimulated conditions.[17] It has been suggested that the proposed downregulation of endothelial ICAM-1 expression facilitates tumor progression by allowing tumor cells to avoid immunosurveillance by circulating lymphocytes. However, there have been several other immunohistochemical studies of the tumor vasculature in which the enhanced expression of endothelial ICAM-1, which resembles an inflammatory phenotype, was detected in breast cancer.[18] Thus, the adhesion molecule expression profile of human LEC remains unclear.

In the present experiment, shear stress stimulation induced ATP release by activating cell surface F1/FO ATP synthase, which resulted in the overexpression of ICAM-1 on human LEC, and, hence, facilitated the ICAM-1-mediated attachment of carcinoma cells to human LEC in the afferent lymph vessels of SLN from breast cancer patients. However, the molecular mechanisms of the shear stress stimulation-mediated activation of cell surface F1/FO ATP synthase on human LEC need to be clarified in future. Thus, except for that obtained in the present study, no information exists regarding the effects of shear stress stimulation on human LEC located near and/or within SLN, particularly with regard to their expression of adhesion molecules, their interactions with carcinoma cells, and their role in the development of a premetastatic microenvironment that encourages carcinoma micrometastasis. Therefore, the present study might be the first to suggest that shear stress stimulation plays a crucial role in the establishment of a pre-metastatic environment within SLN.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The study was financially supported, in part, by Grants-in-Aid for Scientific Research (19209044, 22249052) from the Japanese Ministry of Education, Science, Sports, and Culture and by the Intelligent Surgical Instruments Project of METI (Japan) (2007–2012).

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The authors declare that no competing financial interests exist.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References