Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo.

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models.

In vivo imaging of lymph node lymphangiogenesis by immuno-positron emission tomography.

Recently, our group and others found that cancer and inflammation can induce the expansion of the lymphatic vasculature (lymphangiogenesis) in draining lymph nodes in experimental animal models and in cancer patients (Hirakawa et al., J Exp Med 201:1089-1099, 2005; Qian et al., Cancer Res 66:10365-10376, 2006; Halin et al., Blood 110:3158-3167, 2007; Angeli et al., Immunity 24:203-215, 2006; Dadras et al., Mod Pathol 18:1232-1242, 2005). Since then, a growing number of reports have confirmed the importance of lymph node lymphangiogenesis in tumor metastasis, inflammation resolution, and dendritic cell migration to the lymph nodes (Angeli et al., Immunity 24:203-215, 2006; Hirakawa et al., Blood 109:1010-1017, 2007; Harrell et al., Am J Pathol 170:774-786, 2007; Kataru et al. Blood 113:5650-5659, 2009; Kataru et al., Immunity 34:96-107, 2011; Van den Eynden et al., Clin Cancer Res 13:5391-5397, 2007). Conventionally, lymphangiogenesis in mice is detected by extensive quantitative analysis of lymphatic vessels in stained tissue sections. Here we present a noninvasive methodology that we have recently developed to image lymphangiogenesis in vivo in mice (Mumprecht et al., Cancer Res 70:8842-8851, 2010). This technique is based on the intravenous injection of a radioactively labeled antibody against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), which is almost exclusively expressed on lymphatic vessels (Banerji et al., J Cell Biol 144:789-801, 1999; Preyo et al., J Biol Chem 276:19420-19430, 2001). The accumulation of the injected anti-LYVE-1 antibody in the lymphatic vessels is subsequently visualized by positron emission tomography (PET). Lymphangiogenic lymph nodes emit a stronger radioactive signal than control lymph nodes because they take up more of the radiolabeled anti-LYVE-1 antibody and thus are distinguishable from normal lymph nodes in the PET images.

Inflammation-induced lymph node lymphangiogenesis is reversible.

The extent of lymph node metastasis is a prognostic indicator of disease progression in many malignancies. Current noninvasive imaging technologies for the clinical assessment of lymph node metastases are based on the detection of cancer cells and commonly suffer from a lack of sensitivity. Recent evidence has indicated that the expansion of lymphatic networks (ie, lymphangiogenesis) within tumor-draining lymph nodes might be the earliest sign of metastasis. Therefore, we recently developed a noninvasive imaging method to visualize lymph node lymphangiogenesis in mice using radiolabeled antibodies against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) as well as positron emission tomography (PET). This technique, termed anti-LYVE-1 immuno-PET, was found to be very sensitive in the detection of metastasis to the lymph nodes. However, lymphatic vessel expansion to the lymph nodes can also be induced by inflammation, and it is currently unclear whether such vessel expansion is reversed once inflammation has resolved. Detection of residual inflammation-induced lymph node lymphangiogenesis, thus, might hamper the identification of metastasized lymph nodes. In this study, we therefore used a well-established mouse model of inflammation in the skin to investigate whether lymphatic vessels in the lymph nodes regress on resolution of inflammation. Our data reveal that the lymphatic network indeed regresses on the resolution of inflammation and that we can image this process by anti-LYVE-1 immuno-PET.

In vivo imaging of inflammation- and tumor-induced lymph node lymphangiogenesis by immuno-positron emission tomography.

Metastasis to regional lymph nodes (LN) is a prognostic indicator for cancer progression. There is a great demand for sensitive and noninvasive methods to detect metastasis to LNs. Whereas conventional in vivo imaging approaches have focused on the detection of cancer cells, lymphangiogenesis within tumor-draining LNs might be the earliest sign of metastasis. In mouse models of LN lymphangiogenesis, we found that systemically injected antibodies to lymphatic epitopes accumulated in the lymphatic vasculature in tissues and LNs. Using a (124)I-labeled antibody against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we imaged, for the first time, inflammation- and tumor-draining LNs with expanded lymphatic networks in vivo by positron emission tomography (PET). Anti-LYVE-1 immuno-PET enabled visualization of lymphatic vessel expansion in LNs bearing metastases that were not detected by [(18)F]fluorodeoxyglucose-PET, which is clinically applied to detect cancer metastases. Immuno-PET with lymphatic-specific antibodies may open up new avenues for the early detection of metastasis, and the images obtained might be used as biomarkers for the progression of diseases associated with lymphangiogenesis.

Quantitative imaging of lymphatic function with liposomal indocyanine green.

Lymphatic vessels play a major role in cancer progression and in postsurgical lymphedema, and several new therapeutic approaches targeting lymphatics are currently being developed. Thus, there is a critical need for quantitative imaging methods to measure lymphatic flow. Indocyanine green (ICG) has been used for optical imaging of the lymphatic system, but it is unstable in solution and may rapidly enter venous capillaries after local injection. We developed a novel liposomal formulation of ICG (LP-ICG), resulting in vastly improved stability in solution and an increased fluorescence signal with a shift toward longer wavelength absorption and emission. When injected intradermally to mice, LP-ICG was specifically taken up by lymphatic vessels and allowed improved visualization of deep lymph nodes. In a genetic mouse model of lymphatic dysfunction, injection of LP-ICG showed no enhancement of draining lymph nodes and slower clearance from the injection site. In mice bearing B16 luciferase-expressing melanomas expressing vascular endothelial growth factor-C (VEGF-C), sequential near-IR imaging of intradermally injected LP-ICG enabled quantification of lymphatic flow. Increased flow through draining lymph nodes was observed in mice bearing VEGF-C-expressing tumors without metastases, whereas a decreased flow pattern was seen in mice with a higher lymph node tumor burden. This new method will likely facilitate quantitative studies of lymphatic function in preclinical investigations and may also have potential for imaging of lymphedema or improved sentinel lymph detection in cancer.

Lymphangiogenesis and cancer metastasis.

Metastasis is a characteristic trait of most tumour types and the cause for the majority of cancer deaths. Many tumour types, including melanoma and breast and prostate cancers, first metastasize via lymphatic vessels to their regional lymph nodes. Although the connection between lymph node metastases and shorter survival times of patients was made decades ago, the active involvement of the lymphatic system in cancer, metastasis has been unravelled only recently, after molecular markers of lymphatic vessels were identified. A growing body of evidence indicates that tumour-induced lymphangiogenesis is a predictive indicator of metastasis to lymph nodes and might also be a target for prevention of metastasis. This article reviews the current understanding of lymphangiogenesis in cancer anti-lymphangiogenic strategies for prevention and therapy of metastatic disease, quantification of lymphangiogenesis for the prognosis and diagnosis of metastasis and in vivo imaging technologies for the assessment of lymphatic vessels, drainage and lymph nodes.

Identification of the surface-accessible, lineage-specific vascular proteome by two-dimensional peptide mapping.

The formation of blood vessels (angiogenesis) and of lymphatic vessels (lymphangiogenesis) actively contributes to cancer progression and inflammation. Thus, there has been a quest for identifying the molecular mechanisms that control lymphatic and blood vessel formation and function. Membrane and extracellular matrix proteins can serve as suitable targets for imaging and/or therapeutic targeting; however, conventional proteomic technologies often fail to identify them systematically due to insolubility in water and low abundance of membrane proteins. To circumvent this problem, we applied a gel-free proteomics methodology termed two-dimensional peptide mapping (2D-PM) to cultured blood vascular (BECs) and lymphatic (LECs) endothelial cells. 2D-PM comprises biotinylation of surface-accessible proteins, their selective enrichment, separation by HPLC, and analysis by mass spectrometry. We identified 184 proteins that were specifically or predominantly expressed by LECs and 185 proteins specifically expressed by BECs, whereas 377 additional proteins were equally detected in both cell types. For representative proteins, the differential, lineage-specific expression was confirmed by Western analyses of cultured cells and by differential immunofluorescence analyses of tissue samples. Our results identify the surface-accessible, vascular lineage-specific proteome, and they also reveal 2D-PM as a powerful technology for the large-scale screening of lineage-specific protein expression.