Biomedicine & Pharmacotherapy 68 (2014) 79–86
Available online at
ScienceDirect www.sciencedirect.com
Original article
Aspirin reduces lung cancer metastasis to regional lymph nodes Fumihiro Ogawa a,b, Hideki Amano a, Yoshiya Ito a, Yoshio Matsui b, Kanako Hosono a, Hidero Kitasato c, Yukitoshi Satoh b, Masataka Majima a,* a
Department of Pharmacology, Kitasato University School of Medicine, Kitasato1-15-1, Sagamihara, Kanagawa 252-0374, Japan Department of Thoracic Surgery, Kitasato University School of Medicine, Kitasato1-15-1, Sagamihara, Kanagawa 252-0374, Japan c Department of Microbiology, Kitasato University School of Allied Health Science, 1-15-1, Kitasato, Sagamihara, Kanagawa, 252-0373, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 October 2013 Accepted 11 November 2013
Background: Lung cancer is the main cause of cancer-related death worldwide. The high mortality is probably attributable to early metastasis; however, the mechanism underlying metastasis to regional lymph nodes is still unknown. Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) induces tumor growth and metastasis and is associated with a poor prognosis. The present study investigated the effect of an authentic COX inhibitor, aspirin, on regional lymph node metastasis during the development of lung cancer in mice. Methods: An orthotopic intrapulmonary implantation model based on male C57BL/6 (6–8-weeks-old) mice was used. The lungs were injected with a solution containing Lewis lung carcinoma (LLC) cells overexpressing green fluorescent protein (GFP) and BD Matrigel1. The effect of aspirin on mediastinal lymph node metastasis of LCC cells from the primary injection sites was then examined. Results: The implantation process took approximately 30 s per mouse and operative mortality was 10%. Single pulmonary nodules developed at the implanted site in 95% of animals, and regional mediastinal lymph node metastasis was observed at 14 days post-LLC-GFP cell injection in all mice that formed a primary lung tumor. The mean survival time of mice injected with LLC-GFP cells was 15 3 days (range, 12–22 days). Histopathological analysis revealed that no metastatic tumors developed in the regional mediastinal lymph nodes by Day 10–12 post-LLC-GFP cell injection and no metastasis to distant organs or distant lymph nodes was observed by Day 21 post-injection. Oral administration of aspirin (100 mg/kg, twice a day) after LLC-GFP cell injection inhibited metastasis to the regional lymph nodes, with no significant suppression of primary tumor growth in the lungs. Aspirin treatment led to a significant reduction in mortality (P < 0.0001). Conclusions: The present lymph node metastasis model is useful for evaluating the efficacy of agents that inhibit tumor metastasis to the regional lymph nodes. Aspirin reduced the metastasis of LLC-GFP cells injection to the regional lymph nodes, with a significant reduction in mortality. These findings suggested that COX inhibitors have potential for preventing lymph node metastasis. ß 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Lung cancer Lymph node metastasis Aspirin
1. Introduction Lung cancer is the most common cause of cancer mortality worldwide and non-small-cell lung cancer (NSCLC) accounts for 75–85% of all diagnosed lung cancers. Despite progress in surgical techniques, chemotherapy, and radiotherapy, the 5-year survival rate for patients with lung cancer is only 16% [1]. It is estimated that 226,160 new cases will be diagnosed in 2012 and 160,340 will die from the disease [2]. More than half of patients (56%) with lung cancer have either advanced or metastatic disease at the time of
* Corresponding author. Tel.: +81427788822; fax: +81427787604. E-mail address: [email protected] (M. Majima). 0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.11.006
diagnosis and, even with chemotherapy, the median survival is 1 year or less [3,4]. Tumor recurrence and metastasis are the major causes of treatment failure and death. Metastasis a complex multistep process during which cells must acquire several distinct properties: loss of cell-to-cell adhesion and increased invasiveness, intravasation, and increased survival and proliferation are all prerequisites for the establishment of distant macrometastases [5– 7]. Despite progress in other areas of cancer therapeutics, the complexities of the process mean that cancer metastasis is still poorly understood. Prostanoids, including prostaglandins (PGs), are generated from arachidonic acid via cyclooxygenase (COX) and specific PG synthases. These are the rate-limiting enzymes that regulate PG biosynthesis in various tissues [8–14]. COX-2, an isoform of COX, is
80
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
expressed at sites of inflammation and malignancy. We reported previously that COX-2 and receptor signaling by prostaglandins play crucial roles in lymphangiogenesis during chronic inflammation, secondary lymphedema, and tumor development [8–14]. More precise understanding of the mechanisms underlying lymph node metastases, together with identification of the molecules involved, will improve the prognosis of lung cancer patients. Previous studies show that mice treated with COX inhibitors are resistant to the development of colorectal neoplasia, suggesting that COX inhibitors may be good therapeutic options [15]. The same may be true for humans; epidemiological studies show that non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, a classical and authentic COX inhibitor, reduces the risk of developing several types of cancer [16]. However, the precise role played by COX in regional lymph node metastasis is not clear. Here, we examined regional lymph node metastasis in a mouse model of lung cancer development, and tested the effect of aspirin on regional lymph node metastasis from the primary lung tumor. We found that aspirin inhibited the metastasis of implanted lung cancer cells to regional lymph nodes, resulting in a significant reduction in mortality. These results suggest that aspirin may reduce metastasis of lung cancer cells to the regional lymph nodes and has the potential to extend the lives of lung cancer patients. 2. Materials and methods 2.1. Cell lines LLC cells, originally isolated from C57Bl/6 mice, were cultured at 37 8C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco by Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco by Life Technologies, Grand Island, NY) in a humidified atmosphere containing 5% CO2. LLC cells were purchased from Riken Brc Cell Bank (RBRV-RCB2638; Tsukuba, Japan). 2.2. Retroviral transfection of the GFP gene Murine GPF cDNA was cloned into a deficient retroviral vector, pLEGFP (Clontech by Takara, Tokyo, Japan), and then transfected into PT67 cells (Clontech by Takara, Tokyo, Japan). The cells were then selected by culture with G418 (Roche, Basel, Schweiz). The resulting temporarily infectious recombinant GFP-containing virus was used to infect NIH/3T3 cells, which were then selected with G418 to evaluate the infectious titer. The titer was approximately 1 103 cfu/ml. LLC-GFP cells were infected with the temporarily infectious retroviruses and selected with G418. The selected cells were designed as LLC-GFP. 2.3. Animals Male C57BL/6 mice (6–8-weeks-old; 20–25 g) were obtained from the CLEA Japan (Tokyo, Japan). The mice were maintained at constant humidity (60 5%) and temperature (20 1 8C) and a 12hour light/dark cycle. All animals were provided food and water ad libitum. All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. 2.4. Intrapulmonary implantation LLC-GFP cells (growing in log phase) were suspended in PBS (10 ml; final cell density, 5 104/ml) containing 10 ml of BD Matrigel1 (Becton Dickinson Labware, MA, USA) to prevent air leakage and hemothorax when injected into the lung. The cells were injected into the left lung parenchyma of mice anesthetized with ether [17]. A small skin incision (approximately 5 mm in
length) was made in the chest wall in the left lateral thoracic region. While observing the motion of the left lung through the pleura, a 31 gauge needle attached to a 0.3 ml insulin syringe was directly inserted through the intercostal space into the lung (to a depth of 2–3 mm). After injecting the LLC cells, the skin incision was closed with a surgical suture. Mice were sacrificed at various time points after LLC-GFP cell injection, and the long and short diameters of the primary tumor masses were measured manually. The tumor volume was calculated using ImageJ software (NIH, MD, USA). Lungs harboring a primary tumor nodule and the mediastinal lymph nodes were excised for histological examination and fluorescence microscopy. For the experiment to evaluate the inhibitory effect of NSAIDs, mice were orally administered aspirin (100 mg/kg, twice a day; Sigma-Aldrich, STL, USA) from Day 0 after LLC-GFP injection. At the end of the experiment, the mice were sacrificed with ether and the lungs were resected, fixed with 4% paraformaldehyde (PFA) or 10% formalin. Control (sham operation) mice were injected with LLC cell culture medium, PBS, and BD Matrigel1. 2.5. Fluorescence microscopy Mice were sacrificed as described above, and we observed the status of mediastinal lymph node metastasis examined under a fluorescence microscope (VHX-1000, KEYENCE, Osaka, Japan). The arbitrary units of fluorescence intensity were analyzed using VHX1000 software (KEYENCE) and were compared between the vehicle-treated group and aspirin-treated group. 2.6. Immunohistochemistry Lung tissues were excised and immediately fixed with 4% paraformaldehyde phosphate buffer solution (0.1 mol/L; pH 7.4). After fixation, the tissues were dehydrated in a graded series of ethanol solutions and then embedded in paraffin. Sections (4 mm thick) were cut and mounted on glass slides, deparaffinized with xylene, and placed in acetone at 4 8C. The sections were then blocked with 1% bovine serum albumin-PBS and incubated with Universal DAKO LSAB1 + system-HRP (DAKO, North America, CA, USA) with DAB and Mayer’s hematoxylin solution. Negative control staining was performed by replacing the primary antibodies with 1% bovine serum albumin/PBS. Images were captured using a BX51 microscope (Olympus, Japan). 2.7. Quantitative real-time PCR Each sample of excised tissue was immediately immersed in RNAlater RNA stabilization Reagent (QIAGEN Japan, Tokyo, Japan) and homogenized for 60 seconds at 6000 rpm using a MagNALyser (Roche diagnostics Inc., Mannheim, Germany). Harvested cells were washed three times with PBS and homogenized using a QIA Shredder (QIAGEN Japan, Tokyo, Japan). Total RNA was extracted from the homogenized tissues and cells using the RNeasy Mini Kit (QIAGEN Japan, Tokyo, Japan) and single-stranded cDNA was generated from 1 mg of total RNA by reverse transcription using ReverTra Ace (TOYOBO Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. Quantitative PCR amplification was performed using SYBRR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). The real-time RT-PCR primers for GFP were designed using Primer 3 software (http://primer3.source forge.net) on the basis of data obtained from GenBank. The following primers were used for real-time RT-PCR: GAPDH forward, 50 -ACATCAAGAAGGTGGTGAAGC-30 , and reverse, 50 -AAGGTGGAAGAGTGGGAGTTG-30 ; GFP sense, 50 -ACTACAACAGCCACAACGTCT-30 and antisense, 50 -GGTGT TCTGCTGGTAGTGGTC-30 ; and COX-2 sense, 50 -TGGGTGTGAAGGGAAATAAGG-30 and antisense, 50 -CATCATATTTGAGCCTTGGGG-30 (Sigma-Aldrich).
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
2.8. Statistical analysis Data are expressed as the mean S.E. Comparisons among multiple groups were performed by analysis of variance (ANOVA) followed by Scheffe’s test. Comparisons between two groups were made using Student’s t-test. The correlation between the numbers of metastases was analyzed using the x2 test. P < 0.05 was considered statistically significant. 3. Results 3.1. Survival rate of mice injected with LLC cells plus BD Matrigel1 LLC cells (5.0 104) were orthotopically injected into the left lung through the intercostal space without open thoracotomy. The
81
time taken for the skin incision and intrapulmonary injection of LLC cells was approximately 30 s per mouse. The immediate operative mortality in mice injected with BD Matrigel1 was 10% compared with 80% in the absence of BD Matrigel1 (P < 0.01) (Fig. 1A). The use of BD Matrigel1 prevented death due to pneumothorax. The mean survival time of mice injected with LLCGFP cell was 15 3 days (range, 12–22 days) (Fig. 1B), which was significantly longer than that of sham operation mice (P < 0.0001). Some mice died 14 days post-injection due to hemothorax caused by enlargement of the primary tumor. By Day 21, most of the mice had died. Further analysis revealed tumor metastasis to the regional lymph nodes in the majority of mice. Primary tumors developed at the site of direct implantation in 95% (45/50) of animals. Typical chronological macroscopic images of the LLC tumors are shown in Fig. 1C.
Fig. 1. Regional lymph node metastasis model. A. Survival of mice injected with LLC-GFP in the presence or absence of Matrigel1. The group without Matrigel1 showed a significantly lower survival rate (P < 0.01, n = 20 per group). The Chi-squared test was used to evaluate significant differences. B. Overall survival of mice in the sham (PBS + Matrigel1) and LLC-treated (LLC-GFP + Matrigel1) groups. The survival rate was significantly lower in the LLC-treated group (P < 0.0001; Log-rank test). C. Typical macroscopic images of primary tumors formed over time after LLC-GFP cell injection. The primary tumor showed a gradual increase in size.
82
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
Fig. 2. Time course of regional lymph node metastasis after the injection of LLC plus Matrigel. A. Typical loupe image at Day 10. Typical H&E staining pattern after LLC-GFP cell injection. Bars; 5 mm. PT: primary tumor, LN: lymph node, T: thymus. B. Time course showing the percentage of mice with GFP-positive metastases in the regional lymph nodes. Metastasis was detected 10 days after the LLC-GFP cell injections. On Day 14, most of the LLC-injected mice showed regional/mediastinal lymph node metastasis. C. Typical H&E staining pattern in the regional lymph nodes from mice injected with LLC cells. No LLC cell colonies were detected in the subcapsular regions at Day 10. However, metastases were detected in the subcapsular regions of the lymph nodes at Days 12 and 14 post-LLC cell injection. T: LLC cells.
3.2. Time course of regional lymph node metastasis after the injection of LLC-GFP cells plus BD Matrigel1 Fig. 2A shows a typical loupe image of the primary tumor and a mediastinal lymph node at Day 10 post-LLC-GFP injection. The injected LLC-GFP cells formed colonies in the middle of the left
lung, and LLC-GFP cells metastasized to the regional lymph nodes. Further distant lymph node metastases and other organs’ metastases were noted at later time points. We next examined regional lymph node metastasis by fluorescence microscopy (Fig. 2B). The metastasis rate in mice with a GFP fluorescencepositive regional lymph node was 0% at Day 7 post-LLC-GFP
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
83
Fig. 3. Effect of aspirin on regional lymph node metastasis. A. Temporal changes in LLC-GFP tumor size in the lungs. The results from vehicle-treated mice were compared with those from aspirin-treated mice. Aspirin was given orally (100 mg/kg, twice a day) throughout the experimental period. There was no significant difference in tumor size between the groups. Vehicle-treated group; n = 15. Aspirin-treated group; n = 10. Data are expressed as the mean S.E. ANOVA was used to determine significant differences. B. Typical loupe images after LLC-GFP cell injection, vehicle-only injection (upper panels), and aspirin treatment (lower panels). Bars; 50 mm. Blue arrow; primary tumor, red arrow; mediastinal lymph node, green arrow; thymus. C. Temporal changes in the percentage of mice showing regional lymph node metastasis. Metastasis was compared in the LLC-GFP cell injected, vehicle-treated, and aspirin-treated groups. There was significant difference between the groups. Vehicle group; n = 15. Aspirin-treated group; n = 10 (P < 0.05 Chisquared). D. Overall survival of mice in the vehicle-treated and aspirin-treated groups. Mice in the aspirin-treated group showed improved survival (P < 0.0001; Log-rank test).
84
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
Fig. 4. Effect of aspirin on GFP-positive LLC cell growth and the expression of GFP in the regional lymph nodes. A. Typical Images of lungs and regional lymph nodes taken under a fluorescence microscope. LLC-GFP cells were detected in the regional lymph node 10 days after LCC-GFP injection. Aspirin treatment (100 mg/kg, twice a day) markedly suppressed the accumulation of GFP-positive cells. Bars; 5 mm. PT: primary tumor, LN: lymph node. B. Real-time PCR analysis of GFP expression. GFP mRNA levels were measured in isolated regional lymph nodes by real-time PCR. There was a significant reduction in GFP expression in aspirin-treated mice compared with vehicle-treated mice. Vehicle-treated group, n = 15; Aspirin-treated group, n = 10. Data are expressed as the mean S.E (P < 0.05; ANOVA, overall and P < 0.05; Student’s t-test, each days).
injection. However, the metastasis rates of the GFP fluorescencepositive regional lymph node were increased, 33%, 73%, and 93% at Days 10, 12, and 14, respectively. Fig. 2C shows H&E stained regional lymph nodes. No LLC colonies were observed in the subcapsular sinus of the lymph nodes at Days 3, 5, 7, and 10 post-LLC-GFP cell injection; however, LLC cells were detected in the subcapsular regions at Days 10, 12 and 14 at regional lymph node and no metastatic nodules were observed in distant organs and lymph nodes, including the lung, liver, spleen, adrenal gland, and cervical and subclavian lymph nodes.
Fig. 4A shows typical fluorescence images of lung tissues (with the trachea) from mice bearing GFP-positive LLC cells. We did not detect LLC-GFP cells to fluorescence-positive regional lymph node from vehicle-treated mice at Day 7, but metastases were evident from Day 10 (Fig. 4A; upper panels). These results confirm those obtained after H&E staining (described above). We did not detect any GFP-positive metastases in the aspirin-treated group at any time point (Fig. 4A; lower panels). These results were confirmed by RT-PCR for GFP, which is a much more sensitive technique (Fig. 4B).
3.3. Aspirin treatment reduces regional lymph node metastasis
Here, we used a mouse model of lung cancer [17] generated by injecting LLC-GFP cells directly into the lung. This model has previously been used to examine the characteristics of lung cancer cells [17]; however, it has not been used as a model of lymph node metastasis. This model involves the simple implantation of cancer cells via a small skin incision made at a predetermined site without the requirement for thoracotomy or intubation. The whole implantation process was performed within approximately 30 s and the operative mortality was 10%. Tumors developed at the site of implantation in 95% of animals. Orthotopic models of human lung cancer have been reported by other investigators [18–20]; however, they have several drawbacks, including the need for complicated procedures, such as thoracotomy [20]. Previous
We next examined the effect of an authentic NSAID, aspirin, on regional lymph node metastasis. Aspirin had no significant effect on the size of the primary tumor at Days 0 and 7 post-LLC-GFP injection (Fig. 3A, B). Regional lymph node metastasis was observed in vehicle-treated mice at Days 10, 12, and 14 postLLC-GFP injection; however, this was inhibited in aspirin-treated mice. The percentage of lymph node metastasis-positive mice in the aspirin-treated group was significantly lower than that in the vehicle-treated group (Fig. 3C; P < 0.0001). Furthermore, aspirin treatment led to a significant increase in overall survival (Fig. 3D; P < 0.0001).
4. Discussion
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
studies show that ectopic implantation (i.v., subcutaneous (s.c.), or intra-footpad injections) of lung cancer cells results in the formation of secondary tumors in the lung [21,22]; however, these models may not reflect the clinical course of primary lung cancer. BD Matrigel1 has been used as a standard procedure to facilitate efficient tumor formation. In particular, it facilitates a high take-rate after s.c. implantation of human tumors in athymic mice [23,24]. Injections of cell suspensions containing an indicator dye (without BD Matrigel1) into the lungs caused rapid dispersion of the dye, air leakage, and hemothorax after LLC-GFP cell injection, whereas implantation of cell suspensions containing BD Matrigel1, which gels rapidly at 22–35 8C, resulted in clear localization of the dye at the injection site in the lung. Thus, BD Matrigel1 was essential if this model was to be successful. However, we need to consider the effects of BD Matrigel1 itself. Matrigel can promote tumorigenicity and invasiveness, drug resistance, and induce vascularization [23–27]. As shown in Figs. 1C and 2A, orthotopic implantation of LLC cells suspended in PBS containing BD Matrigel1 resulted in the efficient formation of a solitary tumor nodule. Also, the increase in tumor volume was time-dependent. Similarly, the increase in the rate of mediastinal lymph node metastasis was timedependent (Fig. 2B, C). These results indicate that this model may be a useful basic experimental model for lymph node metastasis during lung cancer development. Prostanoids, including prostaglandins, are generated from arachidonic acid released by COXs; indeed, COXs are the ratelimiting enzymes that regulate PG biosynthesis in various tissues. Two COX isoforms have been identified. COX-1 is constitutively expressed in most tissues and is active in maintaining basal PG levels, which are important for tissue homeostasis. COX-2 is induced in most tissues by various stimuli, and generates proinflammatory PGs during inflammatory responses. Neoplastic tissues, such as human colon cancers, contain high concentrations of PGs. Of these, PGE2 is believed to promote tumor development, tumor growth, and tumor-associated angiogenesis [28]. To investigate the usefulness of this model for evaluating antiinflammatory drugs, we examined the effect of aspirin (an authentic COX inhibitor) on primary tumor growth and lymph node metastasis. Aspirin reduces the risk of developing colorectal cancer, breast cancer, prostate cancer, and esophageal cancer [29– 33]. We found that administration of aspirin twice a day after intrapulmonary injection of LLC-GFP cells inhibited lymph node metastasis without affecting primary tumor growth (Fig. 3A–C). Aspirin also improved overall survival (Fig. 3D). These findings were confirmed by quantitative (Fig. 3C) and fluorescence microscopy (Fig. 4A) analyses and RT-PCR (Fig. 4B). Taken together, these results indicated that aspirin has significant therapeutic potential for the suppression of regional lymph node metastasis and that endogenous PGs may facilitate lymph node metastasis. Further studies using genetically engineered mice and PG receptor and PG biosynthesis systems, such as knockout or transgenic animals, are needed to clarify the mechanisms underlying the role of PGs in cancer metastasis. In conclusion, we demonstrated a simple and reproducible model of tumor metastasis that shows low implantation mortality and a high rate of single nodule development. Thus, this model approximates the clinical picture associated with lung cancer. The present study is the first to show that COX promotes lung cancer metastasis to the regional lymph nodes with animal model. However, further studies are needed to determine the mechanism underlying the relationship between COX/PGs and the microenvironment. This mouse model described herein may be useful for lung cancer research, and research into COX/PGs-related drugs, such as aspirin, as potential suppressors of lymph node metastasis in lung cancer.
85
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We thank Mieko Hamano, Michiko Ogino, and Kyoko Yoshikawa for technical assistance. References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29. [3] Howlader NNA, Krapcho M, Garshell J, Neyman N, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Cho H, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA, editors. SEER Cancer Statistics Review 1975–2010. Cancer Statistics; 2013. [4] Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006;355:2542–50. [5] Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011;331:1559–64. [6] Fidler IJ. The pathogenesis of cancer metastasis: the ‘‘seed and soil’’ hypothesis revisited. Nat Rev Cancer 2003;3:453–8. [7] Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer 2009;9:239–52. [8] Dannenberg AJ, Subbaramaiah K. Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003;4:431–6. [9] Amano H, Hayashi I, Endo H, et al. Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med 2003;197:221–32. [10] Hosono K, Suzuki T, Tamaki H, et al. Roles of prostaglandin E2-EP3/EP4 receptor signaling in the enhancement of lymphangiogenesis during fibroblast growth factor-2-induced granulation formation. Arterioscler Thromb Vasc Biol 2011;31:1049–58. [11] Kashiwagi SHK, Suzuki T, Takeda A, Uchinuma E, Majima M. Role of COX-2 in lymphangiogenesis and restorationof lymphatic flow in secondary lymphedema. Lab Invest 2011;91:12. [12] Katoh H, Hosono K, Ito Y, et al. COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12CXCR4 chemokine systems. Am J Pathol 2010;176:1469–83. [13] Kubo H, Hosono K, Suzuki T, et al. Host prostaglandin EP3 receptor signaling relevant to tumor-associated lymphangiogenesis. Biomed Pharmacother 2010;64:101–6. [14] Majima M, Amano H, Hayashi I. Prostanoid receptor signaling relevant to tumor growth and angiogenesis. Trends Pharmacol Sci 2003;24:524–9. [15] Oshima M, Dinchuk JE, Kargman SL, et al. Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 1996;87:803–9. [16] Thun MJHS, Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 2002;94:252–66. [17] Doki Y, Murakami K, Yamaura T, Sugiyama S, Misaki T, Saiki I. Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice. Brit J Cancer 1999;79:1121–6. [18] McLemore TL, Eggleston JC, Shoemaker RH, et al. Comparison of intrapulmonary, percutaneous intrathoracic, and subcutaneous models for the propagation of human pulmonary and non-pulmonary cancer cell lines in athymic nude mice. Cancer Res 1988;48:2880–6. [19] McLemore TL, Liu MC, Blacker PC, et al. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987;47:5132–40. [20] Wang X, Fu X, Kubota T, Hoffman RM. A new patient-like metastatic model of human small-cell lung cancer constructed orthotopically with intact tissue via thoracotomy in nude mice. Anticancer Res 1992;12:1403–6. [21] Brodt P. Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities. Cancer Res 1986;46:2442–8. [22] Talmadge JE, Fidler IJ. Enhanced metastatic potential of tumor cells harvested from spontaneous metastases of heterogeneous murine tumors. J Natl Cancer Inst 1982;69:975–80. [23] Fridman R, Giaccone G, Kanemoto T, Martin GR, Gazdar AF, Mulshine JL. Reconstituted basement-membrane (matrigel) and laminin can enhance the tumorigenicity and the drug-resistance of small-cell lung-cancer celllines. Proc Natl Acad Sci U S A 1990;87:6698–702. [24] Fridman R, Kibbey MC, Royce LS, et al. Enhanced tumor-growth of both primary and established human and murine tumor-cells in athymic mice after coinjection with matrigel. J Natl Cancer I 1991;83:769–74. [25] Bonfil RD, Vinyals A, Bustuoabad OD, et al. Stimulation of angiogenesis as an explanation of matrigel-enhanced tumorigenicity. Int J Cancer 1994;58:233–9. [26] Ito Y, Iwamoto Y, Tanaka K, Okuyama K, Sugioka Y. A quantitative assay using basement membrane extracts to study tumor angiogenesis in vivo. Int J Cancer 1996;67:148–52.
86
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
[27] Yamamura K, Kibbey MC, Jun SH, Kleinman HK. Effect of Matrigel and laminin peptide YIGSR on tumor growth and metastasis. Semin Cancer Biol 1993;4: 259–65. [28] Greenhough A, Smartt HJ, Moore AE, et al. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377–86. [29] Flossmann E, Rothwell PM. Effect of aspirin on long-term risk of colorectal cancer: consistent evidence from randomised and observational studies. Lancet 2007;369:1603–13. [30] Holmes MD, Chen WY, Li L, Hertzmark E, Spiegelman D, Hankinson SE. Aspirin intake and survival after breast cancer. J Clin Oncol 2010;28:1467–72.
[31] Liu JF, Jamieson GG, Wu TC, Zhu GJ, Drew PA. A preliminary study on the postoperative survival of patients given aspirin after resection for squamous cell carcinoma of the esophagus or adenocarcinoma of the cardia. Ann Surg Oncol 2009;16:1397–402. [32] Rothwell PM, Wilson M, Elwin CE, et al. Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 2010;376:1741–50. [33] Zaorsky NG, Buyyounouski MK, Li T, Horwitz EM. Aspirin and statin nonuse associated with early biochemical failure after prostate radiation therapy. Int J Radiat Oncol Biol Phys 2012;84:e13–7.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Aspirin reduces lung cancer metastasis to regional lymph nodes Fumihiro Ogawa a,b, Hideki Amano a, Yoshiya Ito a, Yoshio Matsui b, Kanako Hosono a, Hidero Kitasato c, Yukitoshi Satoh b, Masataka Majima a,* a
Department of Pharmacology, Kitasato University School of Medicine, Kitasato1-15-1, Sagamihara, Kanagawa 252-0374, Japan Department of Thoracic Surgery, Kitasato University School of Medicine, Kitasato1-15-1, Sagamihara, Kanagawa 252-0374, Japan c Department of Microbiology, Kitasato University School of Allied Health Science, 1-15-1, Kitasato, Sagamihara, Kanagawa, 252-0373, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 October 2013 Accepted 11 November 2013
Background: Lung cancer is the main cause of cancer-related death worldwide. The high mortality is probably attributable to early metastasis; however, the mechanism underlying metastasis to regional lymph nodes is still unknown. Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) induces tumor growth and metastasis and is associated with a poor prognosis. The present study investigated the effect of an authentic COX inhibitor, aspirin, on regional lymph node metastasis during the development of lung cancer in mice. Methods: An orthotopic intrapulmonary implantation model based on male C57BL/6 (6–8-weeks-old) mice was used. The lungs were injected with a solution containing Lewis lung carcinoma (LLC) cells overexpressing green fluorescent protein (GFP) and BD Matrigel1. The effect of aspirin on mediastinal lymph node metastasis of LCC cells from the primary injection sites was then examined. Results: The implantation process took approximately 30 s per mouse and operative mortality was 10%. Single pulmonary nodules developed at the implanted site in 95% of animals, and regional mediastinal lymph node metastasis was observed at 14 days post-LLC-GFP cell injection in all mice that formed a primary lung tumor. The mean survival time of mice injected with LLC-GFP cells was 15 3 days (range, 12–22 days). Histopathological analysis revealed that no metastatic tumors developed in the regional mediastinal lymph nodes by Day 10–12 post-LLC-GFP cell injection and no metastasis to distant organs or distant lymph nodes was observed by Day 21 post-injection. Oral administration of aspirin (100 mg/kg, twice a day) after LLC-GFP cell injection inhibited metastasis to the regional lymph nodes, with no significant suppression of primary tumor growth in the lungs. Aspirin treatment led to a significant reduction in mortality (P < 0.0001). Conclusions: The present lymph node metastasis model is useful for evaluating the efficacy of agents that inhibit tumor metastasis to the regional lymph nodes. Aspirin reduced the metastasis of LLC-GFP cells injection to the regional lymph nodes, with a significant reduction in mortality. These findings suggested that COX inhibitors have potential for preventing lymph node metastasis. ß 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Lung cancer Lymph node metastasis Aspirin
1. Introduction Lung cancer is the most common cause of cancer mortality worldwide and non-small-cell lung cancer (NSCLC) accounts for 75–85% of all diagnosed lung cancers. Despite progress in surgical techniques, chemotherapy, and radiotherapy, the 5-year survival rate for patients with lung cancer is only 16% [1]. It is estimated that 226,160 new cases will be diagnosed in 2012 and 160,340 will die from the disease [2]. More than half of patients (56%) with lung cancer have either advanced or metastatic disease at the time of
* Corresponding author. Tel.: +81427788822; fax: +81427787604. E-mail address: [email protected] (M. Majima). 0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.11.006
diagnosis and, even with chemotherapy, the median survival is 1 year or less [3,4]. Tumor recurrence and metastasis are the major causes of treatment failure and death. Metastasis a complex multistep process during which cells must acquire several distinct properties: loss of cell-to-cell adhesion and increased invasiveness, intravasation, and increased survival and proliferation are all prerequisites for the establishment of distant macrometastases [5– 7]. Despite progress in other areas of cancer therapeutics, the complexities of the process mean that cancer metastasis is still poorly understood. Prostanoids, including prostaglandins (PGs), are generated from arachidonic acid via cyclooxygenase (COX) and specific PG synthases. These are the rate-limiting enzymes that regulate PG biosynthesis in various tissues [8–14]. COX-2, an isoform of COX, is
80
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
expressed at sites of inflammation and malignancy. We reported previously that COX-2 and receptor signaling by prostaglandins play crucial roles in lymphangiogenesis during chronic inflammation, secondary lymphedema, and tumor development [8–14]. More precise understanding of the mechanisms underlying lymph node metastases, together with identification of the molecules involved, will improve the prognosis of lung cancer patients. Previous studies show that mice treated with COX inhibitors are resistant to the development of colorectal neoplasia, suggesting that COX inhibitors may be good therapeutic options [15]. The same may be true for humans; epidemiological studies show that non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, a classical and authentic COX inhibitor, reduces the risk of developing several types of cancer [16]. However, the precise role played by COX in regional lymph node metastasis is not clear. Here, we examined regional lymph node metastasis in a mouse model of lung cancer development, and tested the effect of aspirin on regional lymph node metastasis from the primary lung tumor. We found that aspirin inhibited the metastasis of implanted lung cancer cells to regional lymph nodes, resulting in a significant reduction in mortality. These results suggest that aspirin may reduce metastasis of lung cancer cells to the regional lymph nodes and has the potential to extend the lives of lung cancer patients. 2. Materials and methods 2.1. Cell lines LLC cells, originally isolated from C57Bl/6 mice, were cultured at 37 8C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco by Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco by Life Technologies, Grand Island, NY) in a humidified atmosphere containing 5% CO2. LLC cells were purchased from Riken Brc Cell Bank (RBRV-RCB2638; Tsukuba, Japan). 2.2. Retroviral transfection of the GFP gene Murine GPF cDNA was cloned into a deficient retroviral vector, pLEGFP (Clontech by Takara, Tokyo, Japan), and then transfected into PT67 cells (Clontech by Takara, Tokyo, Japan). The cells were then selected by culture with G418 (Roche, Basel, Schweiz). The resulting temporarily infectious recombinant GFP-containing virus was used to infect NIH/3T3 cells, which were then selected with G418 to evaluate the infectious titer. The titer was approximately 1 103 cfu/ml. LLC-GFP cells were infected with the temporarily infectious retroviruses and selected with G418. The selected cells were designed as LLC-GFP. 2.3. Animals Male C57BL/6 mice (6–8-weeks-old; 20–25 g) were obtained from the CLEA Japan (Tokyo, Japan). The mice were maintained at constant humidity (60 5%) and temperature (20 1 8C) and a 12hour light/dark cycle. All animals were provided food and water ad libitum. All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. 2.4. Intrapulmonary implantation LLC-GFP cells (growing in log phase) were suspended in PBS (10 ml; final cell density, 5 104/ml) containing 10 ml of BD Matrigel1 (Becton Dickinson Labware, MA, USA) to prevent air leakage and hemothorax when injected into the lung. The cells were injected into the left lung parenchyma of mice anesthetized with ether [17]. A small skin incision (approximately 5 mm in
length) was made in the chest wall in the left lateral thoracic region. While observing the motion of the left lung through the pleura, a 31 gauge needle attached to a 0.3 ml insulin syringe was directly inserted through the intercostal space into the lung (to a depth of 2–3 mm). After injecting the LLC cells, the skin incision was closed with a surgical suture. Mice were sacrificed at various time points after LLC-GFP cell injection, and the long and short diameters of the primary tumor masses were measured manually. The tumor volume was calculated using ImageJ software (NIH, MD, USA). Lungs harboring a primary tumor nodule and the mediastinal lymph nodes were excised for histological examination and fluorescence microscopy. For the experiment to evaluate the inhibitory effect of NSAIDs, mice were orally administered aspirin (100 mg/kg, twice a day; Sigma-Aldrich, STL, USA) from Day 0 after LLC-GFP injection. At the end of the experiment, the mice were sacrificed with ether and the lungs were resected, fixed with 4% paraformaldehyde (PFA) or 10% formalin. Control (sham operation) mice were injected with LLC cell culture medium, PBS, and BD Matrigel1. 2.5. Fluorescence microscopy Mice were sacrificed as described above, and we observed the status of mediastinal lymph node metastasis examined under a fluorescence microscope (VHX-1000, KEYENCE, Osaka, Japan). The arbitrary units of fluorescence intensity were analyzed using VHX1000 software (KEYENCE) and were compared between the vehicle-treated group and aspirin-treated group. 2.6. Immunohistochemistry Lung tissues were excised and immediately fixed with 4% paraformaldehyde phosphate buffer solution (0.1 mol/L; pH 7.4). After fixation, the tissues were dehydrated in a graded series of ethanol solutions and then embedded in paraffin. Sections (4 mm thick) were cut and mounted on glass slides, deparaffinized with xylene, and placed in acetone at 4 8C. The sections were then blocked with 1% bovine serum albumin-PBS and incubated with Universal DAKO LSAB1 + system-HRP (DAKO, North America, CA, USA) with DAB and Mayer’s hematoxylin solution. Negative control staining was performed by replacing the primary antibodies with 1% bovine serum albumin/PBS. Images were captured using a BX51 microscope (Olympus, Japan). 2.7. Quantitative real-time PCR Each sample of excised tissue was immediately immersed in RNAlater RNA stabilization Reagent (QIAGEN Japan, Tokyo, Japan) and homogenized for 60 seconds at 6000 rpm using a MagNALyser (Roche diagnostics Inc., Mannheim, Germany). Harvested cells were washed three times with PBS and homogenized using a QIA Shredder (QIAGEN Japan, Tokyo, Japan). Total RNA was extracted from the homogenized tissues and cells using the RNeasy Mini Kit (QIAGEN Japan, Tokyo, Japan) and single-stranded cDNA was generated from 1 mg of total RNA by reverse transcription using ReverTra Ace (TOYOBO Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. Quantitative PCR amplification was performed using SYBRR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). The real-time RT-PCR primers for GFP were designed using Primer 3 software (http://primer3.source forge.net) on the basis of data obtained from GenBank. The following primers were used for real-time RT-PCR: GAPDH forward, 50 -ACATCAAGAAGGTGGTGAAGC-30 , and reverse, 50 -AAGGTGGAAGAGTGGGAGTTG-30 ; GFP sense, 50 -ACTACAACAGCCACAACGTCT-30 and antisense, 50 -GGTGT TCTGCTGGTAGTGGTC-30 ; and COX-2 sense, 50 -TGGGTGTGAAGGGAAATAAGG-30 and antisense, 50 -CATCATATTTGAGCCTTGGGG-30 (Sigma-Aldrich).
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
2.8. Statistical analysis Data are expressed as the mean S.E. Comparisons among multiple groups were performed by analysis of variance (ANOVA) followed by Scheffe’s test. Comparisons between two groups were made using Student’s t-test. The correlation between the numbers of metastases was analyzed using the x2 test. P < 0.05 was considered statistically significant. 3. Results 3.1. Survival rate of mice injected with LLC cells plus BD Matrigel1 LLC cells (5.0 104) were orthotopically injected into the left lung through the intercostal space without open thoracotomy. The
81
time taken for the skin incision and intrapulmonary injection of LLC cells was approximately 30 s per mouse. The immediate operative mortality in mice injected with BD Matrigel1 was 10% compared with 80% in the absence of BD Matrigel1 (P < 0.01) (Fig. 1A). The use of BD Matrigel1 prevented death due to pneumothorax. The mean survival time of mice injected with LLCGFP cell was 15 3 days (range, 12–22 days) (Fig. 1B), which was significantly longer than that of sham operation mice (P < 0.0001). Some mice died 14 days post-injection due to hemothorax caused by enlargement of the primary tumor. By Day 21, most of the mice had died. Further analysis revealed tumor metastasis to the regional lymph nodes in the majority of mice. Primary tumors developed at the site of direct implantation in 95% (45/50) of animals. Typical chronological macroscopic images of the LLC tumors are shown in Fig. 1C.
Fig. 1. Regional lymph node metastasis model. A. Survival of mice injected with LLC-GFP in the presence or absence of Matrigel1. The group without Matrigel1 showed a significantly lower survival rate (P < 0.01, n = 20 per group). The Chi-squared test was used to evaluate significant differences. B. Overall survival of mice in the sham (PBS + Matrigel1) and LLC-treated (LLC-GFP + Matrigel1) groups. The survival rate was significantly lower in the LLC-treated group (P < 0.0001; Log-rank test). C. Typical macroscopic images of primary tumors formed over time after LLC-GFP cell injection. The primary tumor showed a gradual increase in size.
82
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
Fig. 2. Time course of regional lymph node metastasis after the injection of LLC plus Matrigel. A. Typical loupe image at Day 10. Typical H&E staining pattern after LLC-GFP cell injection. Bars; 5 mm. PT: primary tumor, LN: lymph node, T: thymus. B. Time course showing the percentage of mice with GFP-positive metastases in the regional lymph nodes. Metastasis was detected 10 days after the LLC-GFP cell injections. On Day 14, most of the LLC-injected mice showed regional/mediastinal lymph node metastasis. C. Typical H&E staining pattern in the regional lymph nodes from mice injected with LLC cells. No LLC cell colonies were detected in the subcapsular regions at Day 10. However, metastases were detected in the subcapsular regions of the lymph nodes at Days 12 and 14 post-LLC cell injection. T: LLC cells.
3.2. Time course of regional lymph node metastasis after the injection of LLC-GFP cells plus BD Matrigel1 Fig. 2A shows a typical loupe image of the primary tumor and a mediastinal lymph node at Day 10 post-LLC-GFP injection. The injected LLC-GFP cells formed colonies in the middle of the left
lung, and LLC-GFP cells metastasized to the regional lymph nodes. Further distant lymph node metastases and other organs’ metastases were noted at later time points. We next examined regional lymph node metastasis by fluorescence microscopy (Fig. 2B). The metastasis rate in mice with a GFP fluorescencepositive regional lymph node was 0% at Day 7 post-LLC-GFP
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
83
Fig. 3. Effect of aspirin on regional lymph node metastasis. A. Temporal changes in LLC-GFP tumor size in the lungs. The results from vehicle-treated mice were compared with those from aspirin-treated mice. Aspirin was given orally (100 mg/kg, twice a day) throughout the experimental period. There was no significant difference in tumor size between the groups. Vehicle-treated group; n = 15. Aspirin-treated group; n = 10. Data are expressed as the mean S.E. ANOVA was used to determine significant differences. B. Typical loupe images after LLC-GFP cell injection, vehicle-only injection (upper panels), and aspirin treatment (lower panels). Bars; 50 mm. Blue arrow; primary tumor, red arrow; mediastinal lymph node, green arrow; thymus. C. Temporal changes in the percentage of mice showing regional lymph node metastasis. Metastasis was compared in the LLC-GFP cell injected, vehicle-treated, and aspirin-treated groups. There was significant difference between the groups. Vehicle group; n = 15. Aspirin-treated group; n = 10 (P < 0.05 Chisquared). D. Overall survival of mice in the vehicle-treated and aspirin-treated groups. Mice in the aspirin-treated group showed improved survival (P < 0.0001; Log-rank test).
84
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
Fig. 4. Effect of aspirin on GFP-positive LLC cell growth and the expression of GFP in the regional lymph nodes. A. Typical Images of lungs and regional lymph nodes taken under a fluorescence microscope. LLC-GFP cells were detected in the regional lymph node 10 days after LCC-GFP injection. Aspirin treatment (100 mg/kg, twice a day) markedly suppressed the accumulation of GFP-positive cells. Bars; 5 mm. PT: primary tumor, LN: lymph node. B. Real-time PCR analysis of GFP expression. GFP mRNA levels were measured in isolated regional lymph nodes by real-time PCR. There was a significant reduction in GFP expression in aspirin-treated mice compared with vehicle-treated mice. Vehicle-treated group, n = 15; Aspirin-treated group, n = 10. Data are expressed as the mean S.E (P < 0.05; ANOVA, overall and P < 0.05; Student’s t-test, each days).
injection. However, the metastasis rates of the GFP fluorescencepositive regional lymph node were increased, 33%, 73%, and 93% at Days 10, 12, and 14, respectively. Fig. 2C shows H&E stained regional lymph nodes. No LLC colonies were observed in the subcapsular sinus of the lymph nodes at Days 3, 5, 7, and 10 post-LLC-GFP cell injection; however, LLC cells were detected in the subcapsular regions at Days 10, 12 and 14 at regional lymph node and no metastatic nodules were observed in distant organs and lymph nodes, including the lung, liver, spleen, adrenal gland, and cervical and subclavian lymph nodes.
Fig. 4A shows typical fluorescence images of lung tissues (with the trachea) from mice bearing GFP-positive LLC cells. We did not detect LLC-GFP cells to fluorescence-positive regional lymph node from vehicle-treated mice at Day 7, but metastases were evident from Day 10 (Fig. 4A; upper panels). These results confirm those obtained after H&E staining (described above). We did not detect any GFP-positive metastases in the aspirin-treated group at any time point (Fig. 4A; lower panels). These results were confirmed by RT-PCR for GFP, which is a much more sensitive technique (Fig. 4B).
3.3. Aspirin treatment reduces regional lymph node metastasis
Here, we used a mouse model of lung cancer [17] generated by injecting LLC-GFP cells directly into the lung. This model has previously been used to examine the characteristics of lung cancer cells [17]; however, it has not been used as a model of lymph node metastasis. This model involves the simple implantation of cancer cells via a small skin incision made at a predetermined site without the requirement for thoracotomy or intubation. The whole implantation process was performed within approximately 30 s and the operative mortality was 10%. Tumors developed at the site of implantation in 95% of animals. Orthotopic models of human lung cancer have been reported by other investigators [18–20]; however, they have several drawbacks, including the need for complicated procedures, such as thoracotomy [20]. Previous
We next examined the effect of an authentic NSAID, aspirin, on regional lymph node metastasis. Aspirin had no significant effect on the size of the primary tumor at Days 0 and 7 post-LLC-GFP injection (Fig. 3A, B). Regional lymph node metastasis was observed in vehicle-treated mice at Days 10, 12, and 14 postLLC-GFP injection; however, this was inhibited in aspirin-treated mice. The percentage of lymph node metastasis-positive mice in the aspirin-treated group was significantly lower than that in the vehicle-treated group (Fig. 3C; P < 0.0001). Furthermore, aspirin treatment led to a significant increase in overall survival (Fig. 3D; P < 0.0001).
4. Discussion
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
studies show that ectopic implantation (i.v., subcutaneous (s.c.), or intra-footpad injections) of lung cancer cells results in the formation of secondary tumors in the lung [21,22]; however, these models may not reflect the clinical course of primary lung cancer. BD Matrigel1 has been used as a standard procedure to facilitate efficient tumor formation. In particular, it facilitates a high take-rate after s.c. implantation of human tumors in athymic mice [23,24]. Injections of cell suspensions containing an indicator dye (without BD Matrigel1) into the lungs caused rapid dispersion of the dye, air leakage, and hemothorax after LLC-GFP cell injection, whereas implantation of cell suspensions containing BD Matrigel1, which gels rapidly at 22–35 8C, resulted in clear localization of the dye at the injection site in the lung. Thus, BD Matrigel1 was essential if this model was to be successful. However, we need to consider the effects of BD Matrigel1 itself. Matrigel can promote tumorigenicity and invasiveness, drug resistance, and induce vascularization [23–27]. As shown in Figs. 1C and 2A, orthotopic implantation of LLC cells suspended in PBS containing BD Matrigel1 resulted in the efficient formation of a solitary tumor nodule. Also, the increase in tumor volume was time-dependent. Similarly, the increase in the rate of mediastinal lymph node metastasis was timedependent (Fig. 2B, C). These results indicate that this model may be a useful basic experimental model for lymph node metastasis during lung cancer development. Prostanoids, including prostaglandins, are generated from arachidonic acid released by COXs; indeed, COXs are the ratelimiting enzymes that regulate PG biosynthesis in various tissues. Two COX isoforms have been identified. COX-1 is constitutively expressed in most tissues and is active in maintaining basal PG levels, which are important for tissue homeostasis. COX-2 is induced in most tissues by various stimuli, and generates proinflammatory PGs during inflammatory responses. Neoplastic tissues, such as human colon cancers, contain high concentrations of PGs. Of these, PGE2 is believed to promote tumor development, tumor growth, and tumor-associated angiogenesis [28]. To investigate the usefulness of this model for evaluating antiinflammatory drugs, we examined the effect of aspirin (an authentic COX inhibitor) on primary tumor growth and lymph node metastasis. Aspirin reduces the risk of developing colorectal cancer, breast cancer, prostate cancer, and esophageal cancer [29– 33]. We found that administration of aspirin twice a day after intrapulmonary injection of LLC-GFP cells inhibited lymph node metastasis without affecting primary tumor growth (Fig. 3A–C). Aspirin also improved overall survival (Fig. 3D). These findings were confirmed by quantitative (Fig. 3C) and fluorescence microscopy (Fig. 4A) analyses and RT-PCR (Fig. 4B). Taken together, these results indicated that aspirin has significant therapeutic potential for the suppression of regional lymph node metastasis and that endogenous PGs may facilitate lymph node metastasis. Further studies using genetically engineered mice and PG receptor and PG biosynthesis systems, such as knockout or transgenic animals, are needed to clarify the mechanisms underlying the role of PGs in cancer metastasis. In conclusion, we demonstrated a simple and reproducible model of tumor metastasis that shows low implantation mortality and a high rate of single nodule development. Thus, this model approximates the clinical picture associated with lung cancer. The present study is the first to show that COX promotes lung cancer metastasis to the regional lymph nodes with animal model. However, further studies are needed to determine the mechanism underlying the relationship between COX/PGs and the microenvironment. This mouse model described herein may be useful for lung cancer research, and research into COX/PGs-related drugs, such as aspirin, as potential suppressors of lymph node metastasis in lung cancer.
85
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We thank Mieko Hamano, Michiko Ogino, and Kyoko Yoshikawa for technical assistance. References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29. [3] Howlader NNA, Krapcho M, Garshell J, Neyman N, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Cho H, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA, editors. SEER Cancer Statistics Review 1975–2010. Cancer Statistics; 2013. [4] Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006;355:2542–50. [5] Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011;331:1559–64. [6] Fidler IJ. The pathogenesis of cancer metastasis: the ‘‘seed and soil’’ hypothesis revisited. Nat Rev Cancer 2003;3:453–8. [7] Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer 2009;9:239–52. [8] Dannenberg AJ, Subbaramaiah K. Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003;4:431–6. [9] Amano H, Hayashi I, Endo H, et al. Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med 2003;197:221–32. [10] Hosono K, Suzuki T, Tamaki H, et al. Roles of prostaglandin E2-EP3/EP4 receptor signaling in the enhancement of lymphangiogenesis during fibroblast growth factor-2-induced granulation formation. Arterioscler Thromb Vasc Biol 2011;31:1049–58. [11] Kashiwagi SHK, Suzuki T, Takeda A, Uchinuma E, Majima M. Role of COX-2 in lymphangiogenesis and restorationof lymphatic flow in secondary lymphedema. Lab Invest 2011;91:12. [12] Katoh H, Hosono K, Ito Y, et al. COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12CXCR4 chemokine systems. Am J Pathol 2010;176:1469–83. [13] Kubo H, Hosono K, Suzuki T, et al. Host prostaglandin EP3 receptor signaling relevant to tumor-associated lymphangiogenesis. Biomed Pharmacother 2010;64:101–6. [14] Majima M, Amano H, Hayashi I. Prostanoid receptor signaling relevant to tumor growth and angiogenesis. Trends Pharmacol Sci 2003;24:524–9. [15] Oshima M, Dinchuk JE, Kargman SL, et al. Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 1996;87:803–9. [16] Thun MJHS, Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 2002;94:252–66. [17] Doki Y, Murakami K, Yamaura T, Sugiyama S, Misaki T, Saiki I. Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice. Brit J Cancer 1999;79:1121–6. [18] McLemore TL, Eggleston JC, Shoemaker RH, et al. Comparison of intrapulmonary, percutaneous intrathoracic, and subcutaneous models for the propagation of human pulmonary and non-pulmonary cancer cell lines in athymic nude mice. Cancer Res 1988;48:2880–6. [19] McLemore TL, Liu MC, Blacker PC, et al. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987;47:5132–40. [20] Wang X, Fu X, Kubota T, Hoffman RM. A new patient-like metastatic model of human small-cell lung cancer constructed orthotopically with intact tissue via thoracotomy in nude mice. Anticancer Res 1992;12:1403–6. [21] Brodt P. Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities. Cancer Res 1986;46:2442–8. [22] Talmadge JE, Fidler IJ. Enhanced metastatic potential of tumor cells harvested from spontaneous metastases of heterogeneous murine tumors. J Natl Cancer Inst 1982;69:975–80. [23] Fridman R, Giaccone G, Kanemoto T, Martin GR, Gazdar AF, Mulshine JL. Reconstituted basement-membrane (matrigel) and laminin can enhance the tumorigenicity and the drug-resistance of small-cell lung-cancer celllines. Proc Natl Acad Sci U S A 1990;87:6698–702. [24] Fridman R, Kibbey MC, Royce LS, et al. Enhanced tumor-growth of both primary and established human and murine tumor-cells in athymic mice after coinjection with matrigel. J Natl Cancer I 1991;83:769–74. [25] Bonfil RD, Vinyals A, Bustuoabad OD, et al. Stimulation of angiogenesis as an explanation of matrigel-enhanced tumorigenicity. Int J Cancer 1994;58:233–9. [26] Ito Y, Iwamoto Y, Tanaka K, Okuyama K, Sugioka Y. A quantitative assay using basement membrane extracts to study tumor angiogenesis in vivo. Int J Cancer 1996;67:148–52.
86
F. Ogawa et al. / Biomedicine & Pharmacotherapy 68 (2014) 79–86
[27] Yamamura K, Kibbey MC, Jun SH, Kleinman HK. Effect of Matrigel and laminin peptide YIGSR on tumor growth and metastasis. Semin Cancer Biol 1993;4: 259–65. [28] Greenhough A, Smartt HJ, Moore AE, et al. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377–86. [29] Flossmann E, Rothwell PM. Effect of aspirin on long-term risk of colorectal cancer: consistent evidence from randomised and observational studies. Lancet 2007;369:1603–13. [30] Holmes MD, Chen WY, Li L, Hertzmark E, Spiegelman D, Hankinson SE. Aspirin intake and survival after breast cancer. J Clin Oncol 2010;28:1467–72.
[31] Liu JF, Jamieson GG, Wu TC, Zhu GJ, Drew PA. A preliminary study on the postoperative survival of patients given aspirin after resection for squamous cell carcinoma of the esophagus or adenocarcinoma of the cardia. Ann Surg Oncol 2009;16:1397–402. [32] Rothwell PM, Wilson M, Elwin CE, et al. Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 2010;376:1741–50. [33] Zaorsky NG, Buyyounouski MK, Li T, Horwitz EM. Aspirin and statin nonuse associated with early biochemical failure after prostate radiation therapy. Int J Radiat Oncol Biol Phys 2012;84:e13–7.
