Peritumoral Lymphatic Vessel Research Articles

The role of ELK3 to regulate peritumoral lymphangiogenesis and VEGF-C production in triple negative breast cancer cells

Tumor-induced lymphangiogenesis, a major conduit for cancer cell dissemination from the primary tumor site to lymph nodes and beyond, eventually leads to metastasis in cancer patients. Given the recent evidence revealing that the suppression of ELK3 inhibits the metastasis of triple-negative breast cancer cells, we aimed to study the underlying mechanism of impaired metastasis in ELK3-suppressed MDA-MB-231 cells (ELK3 KD) with regard to lymphangiogenesis. We found that the secretome of ELK3 KD cells inhibited tube formation, whereas it promoted the migration and invasion of lymphatic endothelial cells (LECs) in vitro. In vivo analysis revealed that peritumoral lymphatic vessels were not developed around the xenografted tumors of ELK3 KD. We further revealed that the suppression of NF-κB signaling in ELK3 KD was the primary cause of the reduced VEGFC expression. Taken together, we suggest that ELK3 is an upstream regulator of the NF-κB signaling pathway, the inhibition of which leads to the suppression of peritumoral lymphatic vessel development, possibly due to a low VEGFC expression.

Intratumoral and peritumoral lymphatic vessel density both correlate with lymph node metastasis in breast cancer

The status of lymph node involvement is an important prognostic factor for breast cancer. However, the presence of intratumoral lymphatic vessels in primary tumor lesions and the relationship between lymphatic vessel density (LVD) and lymph node metastasis (LNM) have not been firmly established. Therefore, we performed a meta-analysis study to investigate these issues. According to the pre-established inclusion and exclusion criteria, 13 studies, involving 1029 breast cancer patients, were included in this study. Using immunohistochemical staining, intratumoral lymphatic vessels were detected in 40.07% of breast cancer patients (240/599), and peritumoral lymphatics were detected in 77.09% (397/515). All studies demonstrated that peritumoral LVD was higher than intratumoral LVD, with a pooled standard mean difference and 95% confidence interval (95% CI) of 1.75 (1.28 to 2.21). Both intratumoral LVD and peritumoral LVD positively correlated with LNM, with correlation coefficients of 0.14 (95% CI 0.05 to 0.23) and 0.31 (95% CI 0.13 to 0.49), respectively. In summary, our study reports the overall detection rate of intratumoral lymphatics and demonstrates the associations between intratumoral LVD, peritumoral LVD, and LNM in breast cancer. Additionally, controlled studies with a larger number of subjects are needed to establish these relationships.

Multivariate analysis of metastasis‑related risk factors for patients with gastroenteropancreatic neuroendocrine tumors based on clinicopathological and endoscopic features.

Gastroenteropancreatic neuroendocrine tumors (GEP‑NETs) are relatively uncommon. Unfortunately, epidemiological studies on the incidence of GEP‑NETs worldwide have reported a marked increase in the detection of these tumors. Although they often exhibit relatively indolent clinical courses, GEP‑NETs have the potential for lethal progression, especially in patients who present with advanced disease. Early detection and surgical removal is currently the only reliable curative treatment for GEP‑NET patients. The objective of this study was to analyze the clinicopathological characteristics of GEP‑NETs and explore the metastasis‑related risk factors of patients with GEP‑NETs. One hundred and forty‑six patients diagnosed pathologically with GEP‑NETs from January 2001 to January 2015 at the Second Xiangya Hospital of Central South University were retrospectively evaluated. We retrieved and analyzed information concerning clinical characteristics and metastasis‑related risk factors, and used Chi‑square test and logistic regression analysis to analyze the clinicopathological characteristics of GEP‑NETs and explore the association between tumor metastasis and possible related risk factors. The results revealed that the most common clinical manifestations were abdominal pain (n=88), alteration in the character of stool (n=58) and melaena (n=33). Rectum (91/146, 62.3%) and stomach (19/146, 13.0%) were the main sites of metastasis. Both Chi‑square test and logistic regression analysis showed that tumor size (P<0.05), tumor type (P=0.008) and peritumoral lymphatic vessel density(LVD) (P=0.004) were significantly correlated with tumor metastasis. Neither Chi‑square test nor logistic regression analysis indicated that gender (P>0.05), age (P>0.05), tumor location (P>0.05), tumor number (P>0.05), chromaffin granule proteinA[chromograninA(CgA), P>0.05], synaptophysin(Syn, P>0.05) or intratumoral LVD (P>0.05) had a significant correlation with tumor metastasis. Chi‑square test revealed that tumor grade was significantly correlated with tumor metastasis. In conclusion, GEP‑NETs may occur in multiple sites of the digestive system and lack specific clinical manifestations. Tumor size, tumor type, peritumoral LVD, total LVD and tumor grade are metastasis‑related risk factors for GEP‑NET patients.

Abstract B16: Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo

Abstract Background: Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers and is associated with a poorer prognosis and higher rate of distant recurrence compared to receptor positive breast cancer. Due to an absence of effective targeted therapies, cytotoxic chemotherapy remains the standard of care for TNBC patients. However, the aggressive nature of these tumors makes preventing progression to metastatic disease a priority in therapeutic strategy. Inhibition of lymphatic activation represents an attractive approach to reduce peritumoral lymphatic vessel recruitment and attenuate cancer cell invasion to lymph nodes and subsequent metastasis to distant sites. However, little is currently known regarding the role of lymphatics in TNBC and how common therapeutic strategies can influence lymphatic reactivity. We hypothesized that inhibiting lymphatic activation in combination with standard of care chemotherapy will enhance therapeutic response in TNBC. Methods: Our lab has developed a novel 3D tissue-engineered tri-culture system that recapitulates the tumor-lymphatic interface within the breast tumor microenvironment. It consists of a porous tissue culture insert on which we seed human lymphatic endothelial cells (LECs) to form a monolayer on the underside. Atop the insert, we place Collagen I gel with human mammary fibroblasts and human breast cancer cells labeled with fluorescent Cell Tracker dye. After gelation, flow is applied via a pressure head and the system equilibrates overnight (16-18h). Drugs are applied via flow and then flushed from the system with basal media. After 48h from first application, gels are removed, dissociated, and analyzed via flow cytometry for death [Live/Dead and apoptosis (Caspase 3/7)], while the inserts are analyzed via microscopy for invaded cancer cells. Animal experiments were performed according to guidelines set forth by the UVA Animal Care and Use Committee. Twenty female Balb/c mice were implanted with 250,000 4T1 cells and treated with either anti-VEGFR3 antibody or IgG control and docetaxel (n=5 per group) or saline control once tumors were palpable. Tumors were dissected and dissociated once they reached 150 mm3, and analyzed via flow cytometry for stromal cell populations. Lungs were analyzed for metastases by cryosectioning and H&amp;E staining. Results: We used our in vitro system to screen the common chemotherapeutics docetaxel, doxorubicin, 5-FU, and carboplatin across three common TNBC cell lines MDAMB231, HCC1806, and HCC38. We found that the presence of LECs significantly increases TNBC cell survival and invasiveness, shifting EC50s for cell death 10-1000 fold (EC50 shift nonlinear regression, p&amp;lt;0.05 for +/- LECs) and doubling to tripling invasion of cancer cells (analysis by two-way ANOVA, p&amp;lt;0.05). This poor response to therapy can be attenuated by treatment with the VEGFR3 inhibitor MAZ51. In vivo, we have seen that adjuvant anti- VEGFR3 therapy can reduce lymphatic endothelial cell populations in the tumor stroma with and without docetaxel chemotherapy when analyzed by flow cytometry (n=4 per group, p&amp;lt;0.05). Additionally, combination-treated mice showed reduced metastasis to the lung. Conclusion: Together, these data could indicate the effectiveness of anti-VEGFR3 therapy as an addition to chemotherapy to reduce metastatic progression of TNBC by limiting tumor-induced and therapy-induced lymphangiogenesis. Studies are ongoing to optimize and understand the ability of this adjuvant therapeutic strategy to increase therapeutic efficacy in TNBC. Citation Format: Alexandra H. Harris, Jennifer M. Munson. Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B16.

Significance of oral cancer-associated fibroblasts in angiogenesis, lymphangiogenesis, and tumor invasion in oral squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are recognized as a pivotal promoter in cancer initiation and development. However, the role of CAFs in the progression and metastasis of oral squamous cell carcinoma (OSCC) has not been fully elucidated. Lymphatic vessel density (LVD) and microvessel density (MVD) and the expression of α-smooth muscle actin (α-SMA) and matrix metalloproteinase-9 (MMP-9) were evaluated by immunohistochemistry in 86 cases of OSCC. The correlations between α-SMA expression and MMP-9 expression, LVD, MVD, and other clinicopathological parameters were analyzed. In vitro invasion assay was performed to assess the effect of CAFs on the invasion of OSCC cells. We also investigated the effect of CAFs on the angiogenesis and lymphangiogenesis by inoculating CAFs with OSCC cells into nude mice subcutaneously. Positive expression of α-SMA protein was detected in 69.8% of the tumors. Increased α-SMA expression was correlated strongly with enhanced tumor invasion, higher tumor grade, increased risk of recurrence, lymph node involvement, and higher peritumoral lymphatic vessel density and microvessel density (P < 0.05). CAFs induced more cancer cells to invade relative to normal fibroblasts (NFs) (P < 0.05). Compared with co-injection of OSCC cells and NFs or injection of tumor cells alone, co-injection of OSCC cells and CAFs resulted in earlier tumor formation and bigger tumor volume accompanied with increased angiogenesis and lymphangiogenesis (P < 0.05). CAFs may play critical roles in OSCC progression as an inducer of tumor invasion, angiogenesis, and lymphangiogenesis. Therapeutic strategies targeting CAFs against OSCC is promising and need further exploration.

Increased Angiogenesis and Lymphangiogenesis in Metastatic Sentinel Lymph Nodes Is Associated With Nonsentinel Lymph Node Involvement and Distant Metastasis in Patients With Melanoma.

Lymph node angio- and lymphangio-genesis have been shown to play an important role in the premetastatic niche of sentinel lymph nodes. In the current study we have investigated the association of angio- and lympangio-genesis related parameters in metastatic sentinel lymph nodes of patients with melanoma with the presence of nonsentinel and distant organ metastasis. Peritumoral and intratumoral relative blood and lymphatic vessel areas (evaluated by Chalkley method), blood and lymphatic microvessel densities, and the rates of blood and lymphatic vessel proliferation were assessed in primary tumors and sentinel lymph node metastasis of 44 patients with melanoma using CD34/Ki-67 and D240/Ki-67 immunohistochemical double staining. Primary melanoma exhibited significantly higher rate of lymphatic proliferation compared with its lymph node metastasis (P < 0.05), while lymph node metastasis showed significantly higher rate of blood vessel proliferation (P < 0.05). Using multivariate logistic regression model, the rate of peritumoral lymphatic proliferation was inversely associated with positive nonsentinel lymph node status (P < 0.05), whereas the rate of intratumoral blood vessel proliferation was associated with distant organ metastasis (P < 0.05). Using multivariate Cox regression analysis, the rate of intratumoral blood vessel proliferation was also inversely associated with overall survival of patients with melanoma (P < 0.05).

Abstract IA15: Lymphangiogenesis and metastasis of postpartum breast cancer

Abstract Young women diagnosed with breast cancer have a 2-3 fold increased risk of metastatic disease if they are diagnosed within ten years of a completed pregnancy. We find a postpartum diagnosis to be a poor prognostic indicator independent of tumor stage and biologic subtype, leading to the investigation of whether postpartum breast involution is a mediator of metastasis. We show that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. In addition, we show that breast cancer cells exposed to the involuting mammary microenvironment acquire pro-lymphangiogenic properties that contribute to peritumor lymphatic expansion, invasion, and distant metastases. This pro-lymphatic phenotype appears to be due to tumor cells hijacking developmentally-regulated programs normally restricted to the actively involuting mammary gland. Specifically, we find upregulation of lymphangiogenesis with weaning in the normal mammary glands of rodents and humans. Further, in rodent models of postpartum breast cancer, COX-2 inhibition limited to the involution window decreases normal mammary gland lymphangiogenesis, mammary tumor associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Finally, we find that COX-2 inhibition also targets fibroblast activation and collagen production during mammary gland involution, raising the possibility that COX-2 orchestrates a whole tissue response that is supportive of tumor cell escape in the involuting gland. In summary, we show that physiologic COX-2 dependent fibroblast activation, collagen deposition, and lymphangiogenesis are coordinated in the postpartum mammary gland and suggest that tumors evolving in this unique microenvironment acquire enhanced pro-lymphangiogenic and pro-metastatic activities. These data provide further impetus for considering the use of COX-2 inhibitors in the treatment of postpartum breast cancer, and suggest novel prevention strategies targeted to recently pregnant women as well. Citation Format: Pepper J. Schedin, Virginia F. Borges. Lymphangiogenesis and metastasis of postpartum breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA15.

A87: Proliferative activity, lymphatic and blood vessel density in different clinical stages of melanoma

Cutaneous melanoma is one of the most aggressive human neoplasms that can quickly metastasize to regional lymph nodes. Currently, prognosis is determined by measuring tumor thickness but more reliable markers for metastatic spread are urgently needed. It is well known that tumors require a microvasculature development in order to grow and metastasize. Angiogenesis and lymphangiogenesis play an important role not only in the tumor growth, but also in the tumor metastasis. As malignancy is a disorder of cellular growth control, the assessment of cell proliferation rates in tumors has intuitive appeal as a prognostic marker. One such biomarker is Ki-67, a cell cycle dependent protein. Recent reports related to its role as a prognostic factor have been contradictory. The purpose of this study was to investigate lymphangiogenesis, angiogenesis and tumor tissue proliferative activity depending on the clinical stages of melanoma. The objects of the study were the samples of tumor tissue obtained during surgical treatment of cutaneous malignant melanoma. To analyze tumor angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the primary melanomas for the vascular marker CD34, for the lymphatic-specific markers LYVE-1, D2 -40 (Podoplanin) and marker of proliferation – Ki-67. Samples of tumor tissue from 40 patients with IA, IB, IIA, IIB, IIIC melanoma stages were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). Determination of blood vessel volume density revealed its growth an average of 2 times in peritumoral areas. Similar data were obtained about the location lymphatic vessels. Greater content peritumoral blood and lymphatic vessels, then intratumoral could be detected in all stages of melanoma. In addition more significantly greater volume density both intratumoral and peritumoral blood vessels, than lymphatic vessels was found. A greater degree expression of endothelium lymphatic vessels markers Podoplanin, compared with the LYVE-1 was shown. It was noted an increasing volume density of the peritumoral blood and lymphatic vessels in primary tumors with expansion clinical stage melanoma. Three levels of proliferative activity of tumor tissue were determined (low, an average degree and high Ki-67 expression). Results of the analysis have shown, that the high proliferative activity corresponded to significant content of blood and lymphatic vessels. High level of these markers was observed in patients with regional lymph nodes metastases. In recent years there have been appeared publications suggesting that lymphatic vessel density (particularly in a peritumoral location) and lymphatic vessel invasion are predictors of sentinel node metastasis and poorer survival. It was noted, that “larger, carefully conducted and well-reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time-consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas”. In our opinion, one must consider not only the lymphatic vessels density, but also blood vessels, and the tumor tissue proliferative activity. Conclusion This study has shown that the complex markers of lymphangiogenesis (Podoplanin), angiogenesis (CD34) and proliferation (Ki-67) may be predictors of high risk early melanoma metastasis.

Abstract 4115: BNC420 is a novel VEGFR3 selective inhibitor, which unlike the pan-VEGFR inhibitor Sunitinib, suppresses lymphatic metastasis in a model of metastatic melanoma

Abstract The dissemination of cancer cells through the lymphatic system is a well-known route of metastasis. Studies have implicated the hypoxic tumor microenvironment as an instigator of tumor cell invasion and metastatic spread. The use of angiogenesis inhibitors targeting VEGFR2 has been reported in preclinical models to augment metastasis through increased hypoxia within the tumor microenvironment. We have sought to identify a selective VEGFR3 targeting compound that will inhibit metastatic spread through lymphatic dissemination without the concomitant hypoxia induced metastasis effect associated with VEGFR2 targeting. BNC420 is a novel small molecule inhibitor of VEGFR3 phosphorylation that displays significant selectivity over VEGFR2. In phosphorylation detection ELISA based assays BNC420 exhibited a 25 fold greater potency for VEGFR3 over VEGFR2 with IC50 of 30nM and 744nM respectively. We investigated the activity of BNC420 and the pan-VEGFR inhibitor Sunitinib in the B16F10 murine melanoma model. This model involves the intradermal administration of B16F10 melanoma cells in the mouse ear resulting in the formation of a primary lesion, metastatic satellite lesions (in-transit) and regional lymph node metastasis. While Sunitinib slowed the growth of the primary lesion, as expected, it did not inhibit the development of lymph node metastasis and appeared to enhance the development of in-transit satellite lesions by 38% compared to its corresponding vehicle control. In contrast, only 20% of animals treated with BNC420 exhibited in-transit lesions compared to 53% in the corresponding vehicle control. Furthermore, a 50% reduction in the number of metastasis to the draining lymph nodes was seen in BNC420 treated animals. The lymph node metastatic lesions were significantly smaller compared to control and in some instances only detected under microscopic examination. Melanoma lesions in animals treated with BNC420 were devoid of peritumoral lymphatic vessels. These results demonstrate that selective inhibition of VEGFR3 utilising the novel small molecule BNC420 effectively inhibits the development of both in-transit metastatic lesions and lymph node metastasis while being unencumbered by the pro-metastatic effects that can accompany VEGFR2 inhibition through the induction of tumor hypoxia. Citation Format: Annabell Leske, Tina Lavranos, Donna Beaumont, Chloe Brown, Darham Paul, Daniel Inglis, Michaela Scherer, Andrew Harvey, Gabriel Kremmidiotis. BNC420 is a novel VEGFR3 selective inhibitor, which unlike the pan-VEGFR inhibitor Sunitinib, suppresses lymphatic metastasis in a model of metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4115. doi:10.1158/1538-7445.AM2015-4115

Significance of Vascular Endothelial Growth Factor (VEGF)-C and VEGF-D in the Progression of Cutaneous Melanoma.

Induction of tumor lymphangiogenesis by vascular endothelial growth factor (VEGF)-C and VEGF-D promotes metastasis in many human cancers. The aim of this study was to examine the role of VEGF-C and VEGF-D in lymphangiogenesis and lymph node metastasis in patients with cutaneous melanoma. Fifty-four melanoma specimens (18 with lymph node metastasis, 36 nonmetastatic) were investigated by immunostaining for VEGF-C, VEGF-D, and for lymphatic endothelial marker D2-40. VEGF-C and VEGF-D expression was assessed as a percentage and intensity of stained tumor cells, tumor-associated macrophages and fibroblasts. The quantification of lymphangiogenesis was conducted by computer-assisted morphometric analysis. The expressions of both VEGF-C and VEGF-D in tumor cells were significantly higher in lymph node metastatic melanomas compared with nonmetastatic melanomas (P = .015 VEGF-C; P = .005 VEGF-D). There was no statistically significant difference between metastatic and nonmetastatic melanomas regarding the expression of VEGF-C and VEGF-D in macrophages and fibroblasts. Metastatic melanomas showed a significantly higher intratumoral and peritumoral lymphatic vessel density (LVD) compared with nonmetastatic melanomas (P = .000 intratumoral, P = .000 peritumoral). Melanomas with VEGF-C positive tumor cells showed a significantly higher intratumoral and peritumoral LVD compared with VEGF-C negative tumor cells group of melanomas (P = .006 intratumoral, P = .010 peritumoral). VEGF-C expression in macrophages, fibroblasts, as well as VEGF-D expression in tumor cells, macrophages, and fibroblasts, showed no correlation with the intratumoral and peritumoral LVD. Our findings show the significance of VEGF-C in tumor cells in the induction of intratumoral and peritumoral lymphangiogenesis. This study suggests that both VEGF-C and VEGF-D in tumor cells promote lymph node metastasis, and that the immunohistochemical analysis of expression can be a useful tool for predicting clinical behavior of cutaneous melanoma.

Tumor-Associated Lymphatic and Venous Vessels in Medullary Thyroid Carcinomas

Objective: Medullary thyroid carcinomas (MTCs) invade local lymph node through lymphatic vessels and metastasize to distant organs hematogenously and account for a significant mortality. There are possibly increased lymphatic and venous vessels, through which the tumor spreads to lymph nodes and distant organs. Materials and Methods: By immunocytochemical staining for lymphatic and venous vessels, MTC lesions with adjacent normal thyroid and both normal and metastatic lymph nodes were studied for the peritumoral lymphatic and venous vessels, which were morphometrically compared with those of normal thyroid and lymph nodes. Sixteen cases of MTC cases with adjacent thyroid tissues and attached lymph nodes were immunocytochemically stained for lymphatic vessels using lymphatic vessel hyaluronan receptor (LYVE-1) and venous vessels for factor VIII (F-8). The immunostained sections of MTC lesions and metastatic lymph nodes were morphometrically compared for the number and sizes of the vessels with those of normal thyroid tissues and lymph nodes. Results: Significantly increased lymphatic vessels and markedly increased blood vessels were identified in many MTC cases at the peritumoral tissues and metastatic lymph nodes whereas a few lymphatic vessels and no venous vessels were identified in midst of MTCs. The irregular peritumoral lymphatic vessels resembled that of immature lymphatic vessels observed in papillary thyroid carcinomas and increased irregularly, entrapped venous vessels in peritumoral tissues resembled those observed in follicular thyroid carcinomas. Conclusion: The significantly increased lymphatic vessels and markedly increased venous vessels in the peritumoral thyroid tissue support a propensity of MTCs for providing an easy access of tumor cells to both lymphatic spread to the regional lymph nodes and venous spread to distant organs with further tumor spread through metastatic lymph nodes by moderately increased lymphatic and venous vessels.

Lymphangiogenesis in early and advanced gastric cancer: is there any difference?

Gastric cancer (GC) in Poland is on the third place of men's mortality and on the fifth place of women's mortality in malignant neoplasms, and the percentage of diagnosed early GC is less than 20%. In this study, the relationship among lymphatic vessel density, marked with D2-40, expression of vascular endothelial growth factor (VEGF)-C/D, VEGF receptor 3 VEGFR-3, and the stage of GC patient were investigated. This study examined the relationships between the peritumoral lymphatic vessels (PTL) density and intratumoral lymphatic vessels (ITL) density stained immunohistochemically with D2-40/podoplanin, the expression of VEGF-C/D and VEGFR-3, and the stage of 58 GC patients. Lymphatic vessel density measured by D2-40 decreases outside the tumor (PTL) and increases within the tumor (ITL) as the staging grows from I to III, whereas in the case of patients belonging to stage IV group, lymphatic vessel density decreases outside the tumor as well as within the tumor in comparison with the stage III group (not statistically significant). We observed a difference between morphology of the vessels within the tumor (ITLs) and vessels that are located outside the tumor (PTLs). PTLs were enlarged and unsqueezed as opposed to ITLs, which were collapsed. (i) There is no significant correlation between the density of ITL nor PTL marked with D2-40 and the stage of GC. (ii) We did not observe relationship between expression of VEGF-C/D and VEGFR-3 and the stage of GC. (iii) Further studies are needed to fully determine the role of PTL and ITL.

Abstract 4029: BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma

Abstract The role of VEGFR3 in lymphangiogenesis has been well established. Targeting VEGFR3 has been shown to curtail tumor progression mediated via lymphatic dissemination. More recently VEGFR3 was shown to play an important role in the mediation of tumor-induced immune cell tolerance. We have identified BL-011256, a novel inhibitor of VEGFR3 that exhibits 17-fold selectivity over VEGFR2 and a narrow tyrosine kinase inhibition spectrum. A 7-day b.i.d repeat oral dose study showed that BL-011256 is well tolerated in mice. Mouse plasma exposure experiments demonstrated that BL-011256 attains free drug plasma concentration levels that exceed the concentration required for IC50 activity on VEGFR3 in vitro but are considerably lower than the in vitro IC50 required for activity on VEGFR2. In the B16F10 mouse melanoma model, animals bearing melanoma tumors displayed considerably attenuated signs of tumor progression when treated with BL-011256. BL-011256 caused a 70% reduction in primary lesion growth and a 50% reduction in metastasis to the draining lymph node. Furthermore, BL-011256 was active in reducing the number of satellite in-transit metastases. Immunohistochemical whole mount analyses on ears with primary tumor lesions derived from BL-011256 treated and vehicle-treated mice using Lyve-1 for the identification of lymphatic vessels and CD31 for the identification of blood vessels was conducted. Tumors in vehicle-treated mice displayed a peri-tumoral area densely populated by lymphatic vessels. In contrast, tumors derived from BL-011256 treated mice were devoid of peri-tumoral lymphatic vessels. Notably, both vehicle-treated and BL-011256-treated animals displayed similar staining for peritumoral blood vessels, suggesting no effect on blood vessels (this is consistent with no activity on VEGFR2). Furthermore, PK sampling during the last day of dosing in a 14-day dosing schedule demonstrated that there is no compound accumulation during the repeat dosing schedule utilised in the B16F10 tumour efficacy experiment. In conclusion BL-011256 has been identified as a selective inhibitor of VEGFR3 that supresses both primary tumor growth and lymph node metastasis. Citation Format: Annabell Leske, Richard Foitzik, Donna Beaumont, John Bentley, Ylva Bergman, Chloe Brown, Michelle Camerino, Susan Charman, Neil Choi, Melanie De Silva, Matthew Chung, Hendrik Falk, Danny Ganame, Alison Gregg, Julian Grusovin, Andrew Harvey, Catherine Hemley, Ian Holmes, Belinda Huff, Daniel Inglis, Wilhelmus Kersten, Tina Lavranos, Romina Lessene, Gillian Lunniss, Brendon Monahan, Benjamin Morrow, Marica Nikac, George Nikolakopoulos, Dharam Paul, Tom Peat, Justin Ripper, Michaela Scherer, Paul Stupple, Karen White, Ian Street, Gabriel Kremmidiotis. BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4029. doi:10.1158/1538-7445.AM2014-4029

Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer.

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Improved computer-assisted analysis of the global lymphatic network in human cervical tissues

Lymphatic dissemination is a key event in cervical cancer progression and related tumor lymphatic markers are viewed as promising prognostic factor of nodal extension. However, validating such parameters requires an objective characterization of the lymphatic vasculature. Here, we performed a global analysis of the lymphatic network using a new computerized method applied on whole uterine cervical digital images. Sixty-eight cases of cervical neoplasia (12 CIN3, 10 FIGO stage 1A and 46 stage IB1) and 10 cases of normal cervical tissue were reacted with antibodies raised against D2-40, D2-40/p16 and D2-40/Ki67. Immunostained structures were automatically detected on whole slides. The lymphatic vessel density (D2-40), proliferating lymphatic vessel density (D2-40/ki67) and spatial lymphatic distribution in respect to the adjacent epithelium were assessed from normal cervix to early cervical cancer and correlated with lymphovascular space invasion and lymph node status. Prominent lymphatic vessel density and proliferating lymphatic vessel density are detected under the transformation zone of benign cervix and no further increase is noted during cancer progression. Notably, a shift of lymphatic vessel distribution toward the neoplastic edges is detected. In IB1 cervical cancer, although intra- and peritumoral lymphatic vessel density are neither correlated with lymphovascular space invasion nor with lymph node metastasis, a specific spatial distribution with more lymphatic vessels in the vicinity of tumor edges is predictive of lymphatic dissemination. Herein, we provide a new computerized method suitable for an innovative detailed analysis of the lymphatic network. We show that the transformation zone of the benign cervix acts as a baseline lymphangiogenic niche before the initiation of neoplastic process. During cancer progression, this specific microenvironment is maintained with lymphatic vessels even in closer vicinity to tumor cells.

Tumors induce coordinate growth of artery, vein, and lymphatic vessel triads.

BackgroundTumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other.MethodsThe murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels.ResultsEach peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors.ConclusionsImplanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.

Microvascular Invasion of Testicular Nonseminomatous Germ Cell Tumors: Implications of Separate Evaluation of Lymphatic and Blood Vessels

We separately evaluated the lymphatic and blood vascular systems to assess the diagnostic accuracy of microvascular invasion and identify predictive markers for occult metastasis of testicular nonseminomatous germ cell tumors. Tissue samples of 86 patients treated for testicular nonseminomatous germ cell tumors (stage 1 in 48 and stage greater than 1 in 38) were stained using the lymphatic endothelial cell specific marker LYVE-1 and the blood vessel endothelial cell marker von Willebrand factor. We assessed lymph vessel density in LYVE-1 stained sections and blood vessel density in von Willebrand factor stained sections. Lymphovascular invasion in LYVE-1 stained sections and blood vascular invasion in von Willebrand factor stained sections were documented. Parameters were correlated with standard clinicopathological data. Blood vessel density in von Willebrand factor sections was significantly greater than lymphatic vessel density in LYVE-1 sections (p<0.001). Peritumor and nontumor lymphatic vessel density in LYVE-1 sections was associated with metastasis at diagnosis (OR 1.277/U, p=0.020 and OR 1.113/U, p=0.095). Lymphovascular invasion in LYVE-1 sections was significantly associated with metastasis (OR=4.517, p=0.002) but blood vascular invasion in von Willebrand factor sections was only slightly significant (OR 2.261, p=0.071). Only lymphovascular invasion in LYVE-1 stained sections was significantly associated with metastasis in a multiple logistic regression model. Microvascular invasion in hematoxylin and eosin stained sections was not associated with metastasis but microvascular invasion evaluated in LYVE-1 and von Willebrand factor stained sections was associated with metastasis (OR 3.506, p=0.016). Lymphovascular invasion in LYVE-1 stained sections was the most important predictive parameter for metastasis at diagnosis, suggesting greater relevance of the lymphatic system in metastatic dissemination of testicular nonseminomatous germ cell tumors. Vascular endothelial cell specific markers provide higher diagnostic accuracy for microvascular invasion. Our results may impact the current concept of microvascular invasion used for risk stratification of clinical stage 1 testicular nonseminomatous germ cell tumors.

Lymphangiogenesis and Its Correlation with the VEGF Expression and the Sentinel Lymph Node in Cutaneous Melanomas

The aim of the study is to evaluate the density of intratumoral and peritumoral lymphatic vessels in primary cutaneous melanomas and to assess their correlation with the status of sentinel lymph nodes and the VEGF expression in tumor cells and stromal cells. A total of 40 patients were enrolled in the study: the melanomas were radically excised with the extirpation of the sentinel lymph node. The study subjects were divided into two groups: 20 cases with positive and 20 cases with negative sentinel lymph node results. The density of lymphatic vessels was evaluated by the antibody D2-40 and the VEGF expression was investigated in the semiquantitative way. The VEGF expression in melanoma cells and the stromal cells was negative to variable positive at both SLN negative and SLN positive patients in all pT stages. In the group of SLN positive patients, the density of intratumoral lymphatic vessels was low up to moderate, while it was observed to be absent, somewhere on the low level in the group of SLN negative patients. On the other side, the density of peritumoral lymphatic vessels was equally numerous at both SLN negative and SLN positive patients. The lymphatic invasion was found out at 4 SLN positive patients only. The ulceration was chiefly in the group of LN positive patients. The results show that the density of lymphangiogenesis and the intensity of the VEGF expression are considered to be an unreliable predictor of melanoma metastasis to the sentinel lymph node, but the ulceration and the lymphatic invasion can predict the potential for metastasis.

Epigallocatechin-3-Gallate Inhibits Stem-Like Inflammatory Breast Cancer Cells

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes

Recently developed lymphatic-specific immunohistochemical markers can now be utilized to assess intratumoral and/or peritumoral lymphatic vessel density (LVD), to detect lymphatic vessel invasion (LVI) by melanoma cells and to identify lymphatic marker expression in melanoma cells themselves. We systematically reviewed the available evidence for the expression of lymphatic markers as predictors of regional node metastasis and survival in melanoma patients. The currently available evidence suggests that LVD (particularly in a peritumoral location) and LVI are predictors of sentinel node metastasis and poorer survival. Nevertheless, adherence to international guidelines in the conduct and reporting of the studies was generally poor, with wide methodologic variations and heterogeneous findings. Larger, carefully conducted and well-reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time-consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas.