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Supplementary Figure 1 from Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells

Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel–enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells. (Cancer Res 2006; 66(21): 10365-76)

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., ≥2-fold up- or down-regulated, P ≤ 0.05) that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. The tumor LEC profile, validated by immunohistochemical staining, is distinct from that of normal, inflammatory cytokine, or mitogen-activated LEC, characterized by elevated expression of such functionally significant molecules as the tight junction regulatory protein endothelial specific adhesion molecule (ESAM), the transforming growth factor-β coreceptor Endoglin (CD105), the angiogenesis-associated leptin receptor, and the immunoinhibitory receptor CD200, and reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan. Moreover, we show similar induction of ESAM, Endoglin, and leptin receptor within tumor lymphatics in a series of human head and neck and colorectal carcinomas, and uncover a dramatic correlation between ESAM expression and nodal metastasis that identifies this marker as a possible prognostic indicator. These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis. [Cancer Res 2008;68(18):7293–303]

Supplementary Figures 1-8 from A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

Supplementary Figure 2 from Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells

Renal artery injury from blunt abdominal trauma is a rare condition that is typically managed nonoperatively in hemodynamically stable patients. Revascularization can be achieved by stenting or surgical reconstruction of the renal artery. All attempts at revascularization should minimize warm ischemic time. Here, we discuss a patient postmotor vehicle accident who presented to Ryder Trauma Center with intra-abdominal bleeding. He underwent emergency exploratory laparotomy with splenectomy and abdominal packing. Postoperative CT scan revealed a contrast nonenhancing left kidney. The patient then returned to the operating room and underwent in situ renal artery reconstruction after >4 hours of warm ischemia. The patient survived a 2-month hospital course and was discharged home after prolonged in-hospital stay and intensive care treatment. Nuclear medicine scan showed scarring and atrophy of the reattached kidney with 16.3% of overall function attributed to the affected kidney. This case shows that patients with renal artery injury can be managed operatively with arterial reconstruction. Reducing warm ischemic time is critical in preserving kidney function.

The sequence and predicted membrane topology of pannexin1 (Panx1) places it in the family of gap junction proteins. However, rather than forming gap junction channels, Panx1 forms channels in the nonjunctional membrane. Panx1 operates in two distinct open states, depending on the mode of stimulation. The exclusively voltage-gated channel has a small conductance (2+ and the currents through the voltage-activated channel were not altered by Ca2+, Zn2+, Ba2+, or Gd3+. In contrast, the Panx1 channel activated to the large channel conformation by extracellular K+, osmotic stress, or low oxygen was inhibited by the multivalent cations in a dose-dependent way. Thus, monovalent cations activated the Panx1 channel from the closed state to the “large” conformation, while di- and trivalent cations exclusively inhibited this large channel conformation.

Introduction: Boron compounds are being investigated as therapies for dermatologic conditions. Several features of boron chemistry make this element an ideal component in dermatologic treatments. We review the published dermatologically-relevant clinical trials and case studies pertaining to boron compounds.Methods: PubMed was utilized to query terms boron, chemistry, drug, development, dermatology, atopic dermatitis, psoriasis, onychomycosis, tavaborole, AN 2690, crisaborole, and AN 2728. Clinical trials, case studies, animal studies and in vitro studies. Pertaining to atopic dermatitis, psoriasis and onychomycosis were included.Results: Crisaborole 2% topical solution reduced atopic dermatitis lesions by ∼60% when compared to pretreatment baseline. Crisaborole maintains its dose-dependent effect in treatment of psoriasis and significantly reduces psoriatic plaques when compared to controls. Adverse effects were mild, frequency of events varied between studies. Crisaborole was well tolerated when applied to sensitive skin. Topical tavaborole significantly reduced or eliminated onychomycosis with minimal side effects compared to placebo. Tavaborole was effective in treating recalcitrant onychomycosis.Discussion: Boron-based compounds form stable interactions with enzyme targets and are safe medications for the treatment of atopic dermatitis, psoriasis, and onychomycosis. The mild and rare side effects of topical boron-based compounds may make them ideal treatments for individuals with sensitive skin and pediatric populations.

The food dye FD&C Blue No. 1 (Brilliant Blue FCF [BB FCF]) is structurally similar to the purinergic receptor antagonist Brilliant Blue G (BBG), which is a well-known inhibitor of the ionotropic P2X7 receptor (P2X7R). The P2X7R functionally interacts with the membrane channel protein pannexin 1 (Panx1) in inflammasome signaling. Intriguingly, ligands to the P2X7R, regardless of whether they are acting as agonists or antagonists at the receptor, inhibit Panx1 channels. Thus, because both P2X7R and Panx1 are inhibited by BBG, the diagnostic value of the drug is limited. Here, we show that the food dye BB FCF is a selective inhibitor of Panx1 channels, with an IC50 of 0.27 µM. No significant effect was observed with concentrations as high as 100 µM of BB FCF on P2X7R. Differing by just one hydroxyl group from BB FCF, the food dye FD&C Green No. 3 exhibited similar selective inhibition of Panx1 channels. A reverse selectivity was observed for the P2X7R antagonist, oxidized ATP, which in contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels. Based on its selective action, BB FCF can be added to the repertoire of drugs to study the physiology of Panx1 channels. Furthermore, because Panx1 channels appear to be involved directly or indirectly through P2X7Rs in several disorders, BB FCF and derivatives of this “safe” food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohn’s disease, stroke, and injuries to the central nervous system.

Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases.

Pannexin1 (Panx1) participates in several signaling events that involve adenosine triphosphate (ATP) release, including the innate immune response, ciliary beat in airway epithelia, and oxygen supply in the vasculature. The view that Panx1 forms a large ATP release channel has been challenged by the association of a low-conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K + ), Panx1 formed a high-conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K + . The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single-particle electron microscopic analysis revealed that K + stimulated the formation of channels with a larger pore diameter than those formed in the absence of K + . These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.

The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K(+)]o) in a dose-dependent manner. Since increased [K(+)]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K(+)]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K(+) ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen‐induced fibrosarcoma to examine the composition of tumor‐infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4+Foxp3+ regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor‐driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4+ T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen‐driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies., What's new? It is well known that a tumors' microenvironment can impair the anti‐tumor immune response. The culprits are usually assumed to be various suppressor cells and cytokines. In this study, however, the authors found that seemingly innocuous, naïve T cells may also play a significant role—simply by accumulating and possibly out‐competing activated effector cells within the tumor. A better understanding of the signals produced by the tumor microenvironment may allow researchers to alter this T‐cell pool, thus enhancing the immune response.

Changes in the mRNA and protein expression of renin-secreting cells in the juxtaglomerular apparatus (JGA) were examined in the rat following administration of ZENECA ZD8731, an angiotensin II receptor antagonist. Doses of 0 or 90 mg/kg were administered daily by gavage for 26 wk. JGA hypertrophy was apparent in histological sections. Immunohistochemistry demonstrated an increase in the number of renin-containing cells in both the afferent arterioles and the interlobular arteries. Similarly, renin mRNA expression, demonstrated by in situ hybridization, had extended to more proximal segments of the afferent arterioles and was also present in efferent arterioles and interlobular arteries. In conclusion, JGA hypertrophy occurred as a result of antagonism of the angiotensin II receptor. Associated with JGA hypertrophy was increased expression of both renin and renin mRNA, indicative of stimulated renin synthesis caused by an exaggerated pharmacological response of renin-secreting cells to the loss of feedback inhibition by angiotensin II.

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

The field of lymphatic research has benefited enormously from the recent discovery of "marker" proteins that permit not only the identification and quantitation of lymphatic vessels in tissue sections for tumor pathology but also the isolation of primary lymphatic endothelial cells for basic research. This chapter focuses on the use of these markers for the immunohistochemical analysis of lymphangiogenesis in both frozen and paraffin-embedded tissue sections and discusses current protocols and their associated problems.

The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.

The pace of research into the lymphatic system continues to accelerate with the availability of new molecular markers. One such marker, LYVE-1, the lymphatic receptor for the extracellular matrix mucopolysaccharide hyaluronan, has been a key component of many important studies on embryonic and tumour-induced lymphangiogenesis, and continues to be used for the detection and isolation of lymphatic endothelial cells. However, LYVE-1 is interesting in its own right. Being a member of the Link protein family whose only other major hyaluronan receptor is directly involved in leukocyte migration and tumour metastasis, LYVE-1 is already implicated in the trafficking of cells within lymphatic vessels and lymph nodes. The current challenge is to determine the precise roles played by LYVE-1 and other scavenger type receptors in the immune functions of the lymphatics as well as to use LYVE-1 and other markers to investigate the way in which tumours exploit lymphatic vessels for metastasis.

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.

Previous studies of tumor cell-associated procoagulants and fibrinolytic factors have strongly suggested that local thrombin and plasmin generation may be important in tumor growth and dissemination. Given that one central target of both of these serine proteases is fibrin(ogen), a logical extension of this hypothesis is that local fibrin deposition and dissolution may be key determinants of tumor progression. In this paper, the role of fibrin(ogen) and its degradation products in the growth and spontaneous metastasis of Lewis lung carcinoma was directly examined by comparative studies of control and fibrinogen-deficient mice. Fibrinogen deficiency was found to have no effect on the time required for the formation of palpable tumors, tumor angiogenesis, overall tumor architecture, or primary (s.c.) or secondary (pulmonary) tumor growth. However, fibrinogen deficiency markedly reduced the incidence of spontaneous macroscopic metastases in the lung and regional lymph nodes, a process that occurred relatively late in tumor development. Furthermore, a significant quantitative reduction in pulmonary micrometastases was observed in fibrinogen-deficient mice. Quantitative analyses of pulmonary micrometastases in primary tumor-bearing mice indicated that spontaneous showering of tumor cell emboli into the lung was robust, regardless of animal genotype. Hence, our results suggest fibrin(ogen) plays an important role in spontaneous metastasis, facilitating the stable adhesion and/or survival of metastatic emboli after tumor cell intravasation. These studies suggest that therapeutic strategies focusing on hemostatic factors may be effective in controlling solid tumor metastasis, particularly if used for the treatment of micrometastatic disease.

The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of breast cancer. In several human cancers including those of the breast and prostate, the expression of vascular endothelial growth factor C (VEGF-C) is associated with lymph node metastasis. Our study was undertaken to evaluate the effect of VEGF-C on metastasis of poorly invasive, estrogen dependent human MCF-7 breast cancer cells. MCF-7 breast cancer cells transfected with VEGF-C (MCF-7-VEGF-C) were grown as tumors in the mammary fat pads of nude mice implanted with subcutaneous estrogen pellets. Tumor lymphangiogenesis and lymph node metastasis were studied immunohistochemically using antibodies against lymphatic vessel hyaluronan receptor -1 (LYVE-1), VEGF receptor-3 (VEGFR-3), PECAM-1, pan-cytokeratin and estrogen dependent pS2 protein. Overexpression of VEGF-C in transfected MCF-7 cells stimulated in vivo tumor growth in xenotransplanted mice without affecting estrogen responsiveness. The resulting tumors metastasized to the regional lymph nodes in 75% (in 6 mice out of 8, Experiment I) and in 62% (in 5 mice out of 8, Experiment II) of mice bearing orthotopic tumors formed by MCF-7-VEGF-C cells whereas no metastases were observed in mice bearing tumors of control vector-transfected MCF-7 cells (MCF-7-Mock). The density of intratumoral and peritumoral lymphatic vessels was increased in tumors derived from MCF-7-VEGF-C cells but not MCF-7-Mock cells. Taken together, our results show that VEGF-C overexpression stimulates tumor lymphangiogenesis and induces normally poorly metastatic estrogen-dependent MCF-7 tumors to disseminate to local lymph nodes. These data suggest that VEGF-C has an important role in lymph node metastasis of breast cancer even at its hormone-dependent early stage.

How tumors access and spread via the lymphatics is not understood. Although it is clear that dissemination via the blood system involves hemangiogenesis, it is uncertain whether tumors also induce lymphangiogenesis or simply invade existing peritumoral vessels. To address the issue we quantitated tumor lymph vessels in archival specimens of head and neck cancer by immunostaining for the recently described lymphatic endothelial marker LYVE-1, the vascular endothelial marker CD34, and the pKi67 proliferation marker, correlating lymph vessel density and proliferation index with clinical and pathological variables. Discrete "hotspots" of intratumoral small proliferating lymphatics were observed in all carcinomas, and a high intratumoral lymph vessel density was associated with neck node metastases (n = 23; P = 0.027) and an infiltrating margin of tumor invasion (P = 0.046) in the oropharyngeal subgroup. Quantitation of the lymphangiogenic growth factor vascular endothelial growth factor C by real-time PCR and immunohistochemistry revealed higher levels of mRNA in tumor tissue than in normal samples (n = 8; P = 0.017), but no obvious correlation with intratumoral lymphatics. Our results provide new evidence that proliferating lymphatics can occur in human cancers and may in some cases contribute to lymph node metastasis.