Kallistatin inhibits lymphangiogenesis and lymphatic metastasis of gastric cancer by downregulating VEGF-C expression and secretion

Background

Tumor-induced lymphangiogenesis and lymphatic metastasis are predominant during the metastasis of many types of cancers. However, the endogenous inhibitors that counterbalance the lymphangiogenesis and lymphatic metastasis of tumors have not been well evaluated. Kallistatin has been recognized as an endogenous angiogenesis inhibitor.

Methods and results

Our recent study showed for the first time that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Kallistatin expresses anti-lymphangiogenic activity by inhibiting the proliferation, migration, and tube formation of human lymphatic endothelial cells (hLECs). Therefore, the present study focuses on the relationships of changes in kallistatin expression with the lymphangiogenesis and lymphatic metastasis of gastric cancer and its underlying mechanisms. Our results revealed that the expression of kallistatin in cancer tissues, metastatic lymph nodes, and plasma of gastric cancer patients was significantly downregulated and that the plasma level of kallistatin was negatively associated with the phase of lymph node metastasis. Furthermore, treatment with kallistatin recombinant protein decreased LVD and lymph node metastases in the implanted gastric xenograft tumors of nude mice. Mechanically, kallistatin suppressed the lymphangiogenesis and lymphatic metastasis by downregulating VEGF-C expression and secretion through the LRP6/IKK/I?B/NF-?B signaling pathway in gastric cancer cells.

Conclusions

These findings demonstrated that kallistatin functions as an endogenous lymphangiogenesis inhibitor and has an important part in the lymphatic metastasis of gastric cancer.

     

胸段食管癌的淋巴结转移规律及其对确定术后放射治疗靶区范围的价值

目的 分析胸段食管癌淋巴结转移的规律及其影响因素,探讨食管癌术后放疗的靶区范围.方法 收集763例接受根治性切除的胸段食管癌患者的临床病理资料,分析淋巴结转移规律及影响因素.结果 763例胸段食管癌患者共清除淋巴结5846枚,病理证实转移711枚,转移度为12.2%;出现淋巴结转移者297例,转移率为38.9%.胸上段癌淋巴结转移率为28.5%,明显低于胸中段癌(38.8%)和胸下段癌(43.4%).胸上段癌以锁骨上和气管旁淋巴结的转移度和转移率最高.胸中段癌的上行和下行转移均存在,上行主要转移至锁骨上、气管旁和食管旁,下行主要转移至贲门和胃左动脉旁.胸下段癌则主要向食管旁、贲门和胃左动脉旁转移,其中胃左动脉旁的转移度和转移率均显著高于胸上段癌和胸中段癌(均P<0.01).采取左胸单切口的592例患者中,胸上、中、下段癌的淋巴结转移率分别为37.0%、37.9%和41.4%,差异无统计学意义(P=0.715).多因素Logistic回归分析表明,病变长度、浸润深度、脉管瘤栓和远处转移是影响胸段食管癌淋巴结转移的主要因素(均P<0.05).结论 临床上可以根据食管癌的病变长度、浸润深度、脉管瘤栓和远处转移选择需行术后预防照射的患者,根据不同病变部位、不同手术方式及TNM分期,确定术后预防照射的靶区范围.     

Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via reduction of lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.     

Prognostic significance of the ratio between metastatic and dissected lymph nodes (n ratio) in patients with advanced gastric cancer

BACKGROUND AND OBJECTIVES: To determine the prognostic significance of the ratio between metastatic and dissected lymph nodes (n ratio) in gastric cancer patients. METHODS: We retrospectively reviewed 777 advanced gastric cancer patients who had undergone curative gastrectomy at our hospital. RESULTS: The n ratio was significantly greater in cases with a large tumor, undifferentiated tumor, lymphatic vessel invasion, or blood vessel invasion. Furthermore, the n ratio was significantly correlated with the depth of invasion, level of lymph node metastasis, and number of lymph node metastases. The prognosis for gastric cancer patients correlated well with the n ratio. Multivariate analysis indicated that the n ratio, but not the number of lymph node metastases, was an independent prognostic indicator. Moreover, the n ratio was an independent prognostic factor in N1, N2, and N3 patients defined by the Japanese Classification of Gastric Cancer (JCGC). CONCLUSIONS: The n ratio is useful for evaluating the status of lymph node metastasis in gastric cancer. Therefore, the addition of the n ratio to the N (nodal) category defined by the JCGC may be a useful strategy in the N-staging classification of gastric cancer.     

胃癌淋巴结转移相关因素的多元分析

目的 探讨胃癌组织中CD44v6、基质金属蛋白酶－9（MMP－9）、金属蛋白酶抑制剂－1（TIMP－1）和结构型一氧化氮合酶（cNOS)mRNA的表达等因素与胃癌侵袭转移的关系以及在预测胃癌淋巴结转移中的价值。方法 采用Logistic逐步回归的方法,对119例原发性胃癌10项与胃癌淋巴结转移有关的指标进行筛选。结果 胃癌组织中CD44v6表达越强,发生淋巴结转移的机率越高;MMP－9阳性表达而TIMP－1阴性表达者,发生淋巴结转移的机率最高;MMP－9阴性表达而TIMP－1阳性表达者,发生淋巴结转移的机率最低;胃癌血管或淋巴管内皮细胞cNOSmRNA阳性表达者,发生淋巴结转移的机率较高。结论 胃癌组织中CD44v6表达强度、MMP－9和TIMP－1表达失衡情况及胃癌血管或淋巴管内皮细胞cNOSmRNA的表达情况,可作为术前预测患者淋巴结转移的有效指标。     

Evaluation of lymphatic flow pattern using indocyanine green fluorescence imaging in a highly metastatic mouse model

Recently, the feasibility of real‐time indocyanine green (ICG) fluorescence imaging–guided complete mesocolic excision in colon cancer surgery has been demonstrated; however, its application to the evaluation of lymphatic flow in widespread lymph node metastasis is uncertain. This study aimed to evaluate lymphatic flow using the real‐time ICG fluorescence imaging. A mouse model of subcutaneous inoculation of BJMC3879Luc2 cells, which have been demonstrated to highly metastasize to the lymph nodes, was used as an evaluation model. Tumor growth and lymphatic flow were monitored weekly by bioluminescent imaging and near‐infrared (NIR) fluorescence imaging, respectively. After sacrificing the mice, lymph node metastases were evaluated by bioluminescent imaging and histopathology. Lymphatic flows in a model of high lymph node metastasis were evaluated using NIR fluorescence imaging. Pathological metastases of bilateral axillary, femoral, and para‐aortic lymph nodes were detected in all inoculated mice (100%: 5/5). Real‐time NIR fluorescence imaging showed the primary lymphatic vessels staining through the metastatic lymph nodes as before the inoculation of the cancer cells. Hitherto, it has been considered that lymphatic flow was changed using the bypass pathway due to occlusion of the primary lymphatic vessels. In this presented study, real‐time ICG fluorescence imaging showed no changes in lymphatic flow after lymph node metastasis. Our results suggest that real‐time ICG fluorescence imaging may have potential for the guidance of colon cancer surgery in cases of widespread lymph node metastasis.     

Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer.

Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Vascular endothelial growth factor C (VEGF-C) is known to be the only growth factor for the lymphatic vascular system, and its receptor has been identified as Flt4. To clarify the mechanism of lymphatic invasion in cancer, we studied the expression of VEGF-C and flt4 genes in gastric cancer tissues. VEGF-C mRNA was mainly expressed in primary tumors (15 of 32; 47%), but the frequency of VEGF-C mRNA expression was low in normal mucosa (4 of 32; 13%). In primary tumors, there was a significant relationship between VEGF-C and flt4 mRNA expression. In contrast, Flt4 was mainly expressed on the lymphatic endothelial cells but not in cancer cells. A strong correlation was found between VEGF-C expression and lymph node status, lymphatic invasion, venous invasion, and tumor infiltrating patterns. Cancer cells in the lymphatic vessels frequently showed intracytoplasmic VEGF-C immunoreactivity. Furthermore, there was a close correlation between VEGF-C tissue status and the grade of lymph node metastasis. Patients with high expression of VEGF-C protein had a significantly poorer prognosis than did those in low VEGF-C expression group. By the Cox regression model, depth of wall invasion, lymph node metastasis, and VEGF-C tissue status emerged as independent prognostic parameters, and the VEGF-C tissue status was ranked third as an independent risk factor for death. These results strongly suggest that cancer cells producing VEGF-C may induce the proliferation and dilation of lymphatic vessels, resulting in the development of invasion of cancer cells into the lymphatic vessel and lymph node metastasis.     

Tumor metastasis and the lymphatic vasculature

Tumor‐associated lymphatic vessels act as a conduit by which disseminating tumor cells access regional lymph nodes and form metastases there. Lymph node metastasis is of major prognostic significance for many types of cancer, although lymph node metastases are themselves rarely life‐threatening. These observations focus our attention on understanding how tumor cells interact with the lymphatic vasculature, and why this interaction is so significant for prognosis. Tumors interact with the lymphatic vasculature in a number of ways, including vessel co‐option, chemotactic migration and invasion into lymphatic vessels and induction of lymphangiogenesis. Tumor‐induced lymphangiogenesis both locally and in regional lymph nodes has been correlatively and functionally associated with metastasis formation and poor prognosis. The investigation of the molecular regulation of lymphangiogenesis has identified ways of interfering with prolymphangiogenic signaling. Blockade of tumor‐induced lymphangiogenesis in preclinical models inhibits metastasis formation in lymph nodes and often also in other organs, suggesting that blocking the lymphatic route of dissemination might suppress metastasis formation not only in lymph nodes but also in other organs. However, randomized clinical trials that have investigated the efficacy of therapeutic removal of lymph nodes have concluded that lymph node metastases act only as indicators that primary tumors have developed metastatic potential, and do not govern the further spread of metastatic cells. To reconcile these apparently paradoxical observations we suggest a model in which tumor‐induced lymphangiogenesis and lymph node metastasis formation act as indicators that tumors are producing factors that can act systemically to promote metastasis formation in distant organs. © 2009 UICC     

Clinicopathological variables associated with lymph node metastasis in submucosal invasive gastric cancer

Background We aimed to elucidate clinicopathological variables associated with lymph node metastasis of submucosal invasive gastric cancer. Methods Specimens were surgically resected from 201 patients who had primary submucosal gastric cancer. We studied 39 consecutive patients with lymph node metastasis and 162 patients without lymph node metastasis. We compared the following clinicopathological characteristics of the patients in relation to lymph node metastasis: age, sex, tumor size, histology, extent of submucosal invasion, lymphatic and venous invasion, and ulceration of the tumor. Submucosal invasion was divided subjectively into sm1, sm2, and sm3 (representing invasion of the upper-, middle-, and lower-third of the submucosa, respectively). We also studied the relationship between lymph node metastasis of submucosal gastric cancer and immunohistochemistry for p53, Ki67, vascular endothelial growth factor (VEGF), α-fetoprotein, sLe^a, and dendritic cells (DCs). Results In terms of conventional pathological factors, lymph node metastasis in submucosal gastric cancer was related to tumor size (P = 0.002), depth of submucosal invasion (P = 0.001), lymphatic invasion (P < 0.0001), and venous invasion (P = 0.012). Lymph node metastasis in sm1 gastric cancer was significantly related to VEGF expression (P = 0.047). Also, lymph node metastasis in sm3 gastric cancer was significantly correlated with DC expression (P = 0.016). Multivariate analysis showed that tumor size, tumor invasion depth in the submucosal layer, and lymphatic invasion were independent predictors of nodal metastasis in submucosal gastric cancer. Conclusion Conventional pathological factors, such as tumor size, depth of submucosal invasion, and lymphatic invasion, have a significant influence on lymph node metastasis. VEGF expression and DC expression may be helpful predictors of lymph node metastasis in patients with sm1 and sm3 gastric cancer, respectively.     

Cyclooxygenase-2 promotes tumor lymphangiogenesis and lymph node metastasis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer and frequently metastasizes to the cervical lymph nodes, leading to poor survival of patients with OSCC. However, the mechanism of lymph node metastasis is not fully understood. To clarify the molecular mechanism underlying OSCC metastasis to regional lymph nodes, the highly metastatic fluorescent labeled OSCC cell line SAS-LM3 was successfully established allowing us to monitor the progression of lymph node metastases in a non-invasive manner. SAS-LM3 tumors showed increased lymphangiogenesis and elevated expression of VEGF-C, a potent stimulator of lymphangiogenesis, compared to parental SAS tumors. SAS-LM3 showed high expression of cyclooxygenase-2 (COX-2) compared to parental SAS cells and immunohistochemical analysis demonstrated intense COX-2 expression at the primary site. Inactivation of COX-2 by knockdown or the COX-2 inhibitor NS-398 decreased VEGF-C expression. Administration of COX-2 inhibitor NS-398 in SAS-LM3 tumor-bearing mice suppressed tumor lymphangiogenesis and lymphatic metastases. Collectively, our results indicate that COX-2 promotes tumor lymphangiogenesis and lymph node metastasis of OSCC. COX-2 ablation holds promise as a potential therapeutic approach for lymph node metastasis in OSCC.     

早期胃癌的淋巴结转移规律及预后分析

目的 分析早期胃癌的临床病理特征与预后之间的关系及早期胃癌的淋巴结转移规律.方法 对1994年1月～2005年10月手术治疗并有完整资料的255例早期胃癌的临床病理学资料进行回顾性分析.结果 255例患者的总5年生存率为91.4%.单因素分析显示,肿瘤浸润深度、脉管瘤栓和区域淋巴结转移与患者术后生存率有关;而性别、年龄...     

Determining of metastatic lymph node ratio in patients who underwent D2 dissection for gastric cancer

The purpose of this study was to determine outcome of the ratio of metastatic lymph nodes to the total number of dissected lymph nodes (MLR) in patients with gastric cancer. We retrospectively analyzed 111 patients who underwent D₂ lymph node dissection. The prognostic factors including UICC/AJCC TNM classification and MLR were evaluated by univariate and multivariate analysis. The MLR was significantly higher in patients with a larger tumor, lymphatic vessel invasion, blood vessel invasion and perineural invasion, and advanced stage. Moreover, the MLR was significantly associated with the depth of invasion and the number of lymph node metastasis. The univariate analysis revealed for overall survival (OS) that stage of disease, lymphatic vessel invasion, blood vessel invasion, perineural invasion, lymph node metastasis (UICC/AJCC pN stage) and MLR were relevant prognostic indicators. Furthermore, both UICC/AJCC pN stage and MLR were detected as prognostic factor by multivariate analysis, as was perineural invasion. Our results indicated that MLR and UICC/AJCC pN staging system were important prognostic factors for OS of patients with D₂ lymph node dissection in gastric cancer in a multivariate analysis. MLR may be useful for evaluating the status of lymph node metastasis in gastric cancer.     

Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling

BACKGROUND: Vascular endothelial growth factor C (VEGF-C) stimulates tumor lymphangiogenesis (i.e., formation of lymphatic vessels) and metastasis to regional lymph nodes by interacting with VEGF receptor 3 (VEGFR-3). We sought to determine whether inhibiting VEGFR-3 signaling, and thus tumor lymphangiogenesis, would inhibit tumor metastasis. METHODS: We used the highly metastatic human lung cancer cell line NCI-H460-LNM35 (LNM35) and its parental line NCI-H460-N15 (N15) with low metastatic capacity. We inserted genes by transfection and established a stable N15 cell line secreting VEGF-C and a LNM35 cell line secreting the soluble fusion protein VEGF receptor 3-immunoglobulin (VEGFR-3-Ig, which binds VEGF-C and inhibits VEGFR-3 signaling). Control lines were transfected with mock vectors. Tumor cells were implanted subcutaneously into severe combined immunodeficient mice (n = 6 in each group), and tumors and metastases were examined 6 weeks later. In another approach, recombinant adenoviruses expressing VEGFR-3-Ig (AdR3-Ig) or beta-galactosidase (AdLacZ) were injected intravenously into LNM35 tumor-bearing mice (n = 14 and 7, respectively). RESULTS: LNM35 cells expressed higher levels of VEGF-C RNA and protein than did N15 cells. Xenograft mock vector-transfected LNM35 tumors showed more intratumoral lymphatic vessels (15.3 vessels per grid; 95% confidence interval [CI] = 13.3 to 17.4) and more metastases in draining lymph nodes (12 of 12) than VEGFR-3-Ig-transfected LNM35 tumors (4.1 vessels per grid; 95% CI = 3.4 to 4.7; P<.001, two-sided t test; and four lymph nodes with metastases of 12 lymph nodes examined). Lymph node metastasis was also inhibited in AdR3-Ig-treated mice (AdR3-Ig = 0 of 28 lymph nodes; AdLacZ = 11 of 14 lymph nodes). However, metastasis to the lungs occurred in all mice, suggesting that LNM35 cells can also spread via other mechanisms. N15 tumors overexpressing VEGF-C contained more lymphatic vessels than vector-transfected tumors but did not have increased metastatic ability. CONCLUSIONS: Lymph node metastasis appears to be regulated by additional factors besides VEGF-C. Inhibition of VEGFR-3 signaling can suppress tumor lymphangiogenesis and metastasis to regional lymph nodes but not to lungs.     

Suppression of VEGFR-3 signaling inhibits lymph node metastasis in gastric cancer

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF-C or VEGF-D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR-3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR-3 and other specific lymphatic markers, LYVE-1 and prox-1. Then we systemically administered anti-VEGFR-3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE-1-positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR-3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.     

Development of new spontaneous metastatic heterotopic model of lewis lung carcinoma imaged by GFP expression

Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups ?sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases.     

Preventive effect of matrix metalloproteinase inhibitor, R-94138, in combination with mitomycin C or cisplatin on peritoneal dissemination of human gastric cancer cell line TMK-1 in nude mice.

R-94138, a matrix metalloproteinase inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK-1. When the supernatant of a co-culture of TMK-1 cells and human normal fibroblast cells was subjected to gelatin zymography, it was clear that the protein expression of MMP-2 had been inhibited by R-94138. When TMK-1 was injected intraperitoneally (i.p.) into nude mice at 5 x 10(5) cells/body, the resulting peritoneal dissemination mimicked clinical carcinomatous peritonitis. When the maximum tolerated dose of mitomycin C (MMC) or cisplatin (DDP) was given 12 h after the tumor inoculation, peritoneal dissemination was completely inhibited, while the effect of R-94138 was limited when it was given i.p. at a dose of 20 mg/kg in a schedule of q.d. x 5 starting 12 h after tumor injection. MMC and DDP also suppressed peritoneal dissemination when they were administered 1 week after the tumor inoculation at a single dose of 2 and 3 mg/kg i.p., respectively. R-94138 inhibited peritoneal dissemination when it was administered i.p. at a dose of 30 mg/kg in a schedule of q.d. x 5 starting from 1 week after tumor injection. The combination of MMC and R-94138 increased the preventive effect on peritoneal dissemination. R-94138 seems to be a promising candidate to prevent peritoneal dissemination of gastric cancer.     

胃癌原发灶和淋巴结转移灶HER2表达的临床意义

目的 观察HER2在胃癌原发灶和淋巴结转移灶中的表达及其临床意义.方法 选取胃癌患者140例,其中淋巴结转移94例.采集胃癌患者的原发灶、淋巴结转移灶及癌旁组织,采用免疫组织化学法(Elivision)方法检测3种组织中HER2蛋白表达情况.结果 140例胃癌原发灶组织中HER2蛋白阳性表达与胃癌TNM分期、浸润深度及淋巴结转移有关(P<0.05),而与患者性别、年龄和分化程度无关(P>0.05),癌旁组织中HER2表达与性别、年龄、分化程度、TNM分期、浸润深度及淋巴结转移均无关(P>0.05);HER2蛋白在胃癌原发灶、淋巴结转移灶中表达水平均高于癌旁组织,差异有统计学意义,而94例淋巴结转移灶和对应的胃癌原发灶中HER2表达的差异无统计学意义;94例有淋巴结转移的患者淋巴结转移灶与原发灶HER2表达一致率为89.4%,两类标本HER2表达状态具有一致性(Z=6.386,P<0.001).结论 胃癌HER2蛋白的阳性表达与胃癌TNM分期、浸润深度及淋巴结转移有关,提示HER2的表达与胃癌的浸润转移有关;胃癌原发灶和淋巴结转移灶HER2的表达具有较好的一致性,患者在不能获取原发病灶的情况下,检测转移灶中HER-2可能为靶向治疗的选择提供依据,为晚期胃癌患者带来希望.