TGFß counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFß in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFß Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

Inhibition of VEGFR-3 activation in tumor-draining lymph nodes suppresses the outgrowth of lymph node metastases in the MT-450 syngeneic rat breast cancer model.

For many types of human cancer, the expression of vascular endothelial growth factor-C (VEGF-C) correlates with enhanced tumor-associated lymphatic vessel density, metastasis formation and poor prognosis. In experimental animals, VEGF-C produced by primary tumors can induce lymphangiogenesis within and/or at the periphery of the tumor, and promotes metastasis formation. Tumor-induced lymphangiogenesis is therefore thought to expedite entry of tumor cells into the lymphatic vasculature and their trafficking to regional lymph nodes, thereby fostering metastatic dissemination. Tumour-produced VEGF-C can also drain to the regional lymph nodes and induce lymphangiogenesis there. Whether this activity promotes metastasis formation remains unclear. To address this issue we manipulated VEGF-C activity and VEGFR-3 activation in the lymph nodes draining syngeneic rat breast cancers using intra-dermal delivery of either recombinant VEGF-C or VEGFR-3 blocking antibodies to induce or suppress lymph node lymphangiogenesis, respectively. Recombinant VEGF-C induced lymph node lymphangiogenesis, but was not sufficient to promote metastasis formation by poorly metastatic NM-081 breast tumours. Conversely, inhibition of lymph node lymphangiogeneis induced by highly metastatic MT-450 breast tumours suppressed the outgrowth of lymph node metastases, but not the initial colonization of the lymph nodes. Lung metastasis was also not affected. We conclude that tumor-derived VEGF-C draining to regional lymph nodes promotes the outgrowth of lymph node metastases. VEGF-C may induce lung metastasis independently of its effects on lymph node metastasis.

Microneedle delivery: clinical studies and emerging medical applications.

The concept of microneedle drug delivery was described three decades ago; however, effective clinical demonstration has only occurred within the past 10-15 years. Substantial progress in microneedle design and fabrication including extensive in vitro, ex vivo, and in vivo preclinical evaluation with various drugs, vaccines and other agents has transpired over the last decade. In contrast with this large volume of preclinical data, there are relatively few published microneedle clinical studies. To date, the clinical investigative focus has included testing to reduce dermal barrier properties and enhance transdermal delivery; evaluation of enhanced vaccine antigenicity, including development of the first commercial microneedle product for intradermal influenza vaccination; evaluation of altered microneedle protein pharmacokinetics and pharmacodynamics, especially for insulin; and evaluation of the pain and other perceptions associated with microneedle usage. This review summarizes the various aspects of microneedle clinical evaluation to date and identifies areas requiring further clinical evaluation.

Microneedle-based intradermal delivery enables rapid lymphatic uptake and distribution of protein drugs.

PURPOSE: The purpose of this research was to examine the pharmacokinetics (PK) of drug uptake for microneedle-based intradermal (ID) delivery of several classes of protein drugs compared to standard subcutaneous (SC) administration. METHODS: Systemic absorption kinetics of various proteins were analyzed following microneedle-based ID delivery and standard injection methods in the swine model. Comparative PK data were determined using standard non-compartmental techniques based on blood serum levels. RESULTS: Delivery of proteins using microneedles resulted in faster systemic availability, measured via t(max,) and increased maximal drug concentration, C(max,) over SC delivery for all proteins tested. Some agents also exhibited increased bioavailability for the ID route. Imaging studies using reporter dyes showed rapid lymphatic-mediated uptake. CONCLUSIONS: Microneedle delivery is applicable to a wide variety of protein drugs and is capable of effective parenteral administration of therapeutic drug dosages. This delivery route alters absorption kinetics via targeting a tissue bed better perfused with lymphatic and blood vessels than the SC space. Microneedle delivery may afford various advantages, including a robust method to increase the absorption rate and bioavailability of proteins that have been challenging to deliver at therapeutic levels or with physiologically relevant profiles.

Cellular magnetic resonance imaging: in vivo imaging of melanoma cells in lymph nodes of mice.

Metastasis is responsible for most deaths due to malignant melanoma. The clinical significance of micrometastases in the lymph is a hotly debated topic, but an improved understanding of the lymphatic spread of cancer remains important for improving cancer survival. Cellular magnetic resonance imaging (MRI) is a newly emerging field of imaging research that is expected to have a large impact on cancer research. In this study, we demonstrate the cellular MRI technology required to reliably image the lymphatic system in mice and to detect iron-labeled metastatic melanoma cells within the mouse lymph nodes. Melanoma cells were implanted directly into the inguinal lymph nodes in mice, and micro-MRI was performed using a customized 1.5-T clinical MRI system. We show cell detection of as few as 100 iron-labeled cells within the lymph node, with injections of larger cell numbers producing increasingly obvious regions of signal void. In addition, we show that cellular MRI allows monitoring of the fate of these cells over time as they develop into intranodal tumors. This technology will allow noninvasive investigations of cellular events in cancer metastasis within an entire animal and will facilitate progress in understanding the mechanisms of metastasis within the lymphatic system.