Summary In vivo imaging technologies drive the development of improved cancer therapies by revealing critical aspects of the complex pathophysiology of solid tumors in small animal models[1]. The abnormal vascular function, which predicts tumor malignant potential and presents broad barriers to effective treatment, has been studied at the subcellular size scale using multiphoton (MP) microscopy [2], and at significantly larger size scales using ultrasound, µCT and µMRI[3–5]. However, limited in vivo imaging approaches exist to study the vascular function at the network level, i.e., with sufficient resolution to discern smaller vessels while maintaining a field of view and penetration depth large enough to reveal interconnectivity and inhomogeneities across the tumor and surrounding tissue. One promising technology operating at this size scale is optical frequency domain imaging (OFDI) using Doppler-methods to detect blood flow.

Inhibition of lymphangiogenesis prevents lymphatic cancer metastasis, whereas induction of lymphangiogenesis alleviates lymphedema in experimental animal models. The number of known molecular players that govern the formation and function of the microlymphatic system is growing. Here, we review the role of nitric oxide within the regulation of lymphatic formation and function and point out key unanswered questions for its translation into clinical therapy.

Functional interactions between the initial and collecting lymphatics, as well as the molecular players involved, remain elusive. In this study, we assessed the influence of nitric oxide (NO) on lymphatic fluid velocity and flow, using a mouse tail model that permits intravital microscopy and microlymphangiography. We found that NO synthase (NOS) inhibition decreased lymphatic fluid velocity in the initial lymphatics, without any effect on their morphology. Using the same model, we found a similar effect in eNOS−/− mice and in mice treated with a selective endothelial NOS (eNOS) inhibitor. Next, we uncoupled the superficial initial lymphatics from the deeper collecting lymphatics by ligating the latter and found that lymphatic fluid velocity in NOS-inhibited mice became equal to that in control animals. Surprisingly, lymphatic fluid velocity was significantly increased after ligating the collecting lymphatics, and there was a concomitant increase in injection rate and mean lymphatic vessel diameter. Our results provide the first in vivo evidence that eNOS affects function of the whole microlymphatic system and that it is regulated via the collecting lymphatics.

Vascular endothelial growth factor (VEGF)-C is known to induce hyperplasia in normal murine lymphatics and in peritumor lymphatics. Here, we examine the function of these hyperplastic peritumor lymphatics. Microlymphangiography of B16F10 melanomas growing in the murine dorsal skinfold chamber showed that the number of functional, draining lymphatics in the peritumor tissue of VEGF-C-overexpressing tumors was significantly greater than that in mock-transduced tumors (9.5 ± 1.0 versus 6.3 ± 0.4; n = 6; P < 0.05). Forty percent of functional lymphatics associated with VEGF-C-overexpressing tumors contained proliferating lymphatic endothelial cells. Surprisingly, these new, functional lymphatic vessels displayed a retrograde draining pattern, which indicates possible dysfunction of the intraluminal valves of these vessels.

We read with interest the article by Maula et al . [(1)][1] , showing that CD44 expression of the primary tumor is an independent prognostic parameter for decreased disease-free survival in patients with HNSCC. [1][2] However, the authors also suggest, as the title states, that intratumor lymphatic

Cancer cells escape a tumor by two primary routes-blood vessels and lymphatic vessels-to establish distant metastases.Thus, it seems reasonable to hypothesize that blocking the growth of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) will inhibit hematogenic and lymphogenic metastases, respectively.An impressive array of preclinical studies has demonstrated prevention and suppression of hematogenic metastases by antiangiogenic and antivascular approaches.Whether antilymphangiogenic and antilymphatic approaches will yield similar results for lymphogenic metastases remains to be seen.Both vascular endothelial growth factor (VEGF)-C and VEGF-D induce angiogenesis (1,2) and lymphangiogenesis (1,(3)(4)(5) in tumors and are associated with lymphogenic metastasis in a variety of human tumors (6).Last year Stacker et al. (1) presented the first direct evidence for the prevention of lymphatic metastasis from a tumor grown in the mammary fat pad by blocking VEGF-D.In this issue of the Journal, He et al. (7) report similar findings in subcutaneously grown tumors that lack VEGF-D by blocking VEGF-C.Although Stacker et al. (1) used a blocking antibody against VEGF-D, He et al. ( 7) used a receptor-antibody fusion protein (VEGFR-3-Ig fusion protein) that can trap both VEGF-C and VEGF-D.The receptor-antibody was generated in vivo by cancer cells engineered to secrete soluble VEGFR-3-Ig or by the liver infected by an adenovirus expressing VEGFR-3-Ig.In both studies, treatment was initiated within a day after tumor implantation, before the primary tumor became established, and lymph nodes were examined for metastatic lesions 4-6 weeks later.Blocking VEGF-C or VEGF-D suppressed lymphangiogenesis associated with the primary tumor and regional lymph node metastasis.These are exciting and timely findings that raise many important questions about the biology and potential treatment of lymphatic metastases.First, how do VEGF-C and VEGF-D alter tumor biology to promote lymphatic metastasis?The current study used an ectopic (subcutaneous) model of lung cancer and found an association between lymphatic vessel density (measured as lymphatic vessel endothelial hyaluronan receptor [LYVE]-1-positive structures) and lymph node metastases in one of two tumor lines.Another recent study (4) used orthotopically grown tumors engineered to overexpress VEGF-C and found an increased diameter of functional lymphatic vessels in the tumor margin and a greater number of lymph node metastases compared with mocktransduced controls.Thus, by increasing the surface area of lymphatic vessels, VEGF-C and VEGF-D increase the opportunity for metastatic spread through lymphatic vessels.VEGF-C and VEGF-D may also serve as a survival factor for endothelial cells of newly formed or co-opted lymphatic vessels in a tumor (5).Furthermore, He et al. (7) show that trapping VEGF-C only inhibits lymphatic metastasis without affecting hematogenic

Lymphatic metastasis contributes to mortality from solid tumors. Whether metastasizing cancer cells reach lymph nodes via intratumor lymphatic vessels is unknown. Here, we examine functional lymphatics associated with mouse tumors expressing normal or elevated levels of vascular endothelial growth factor-C (VEGF-C), a molecule that stimulates lymphangiogenesis. Although VEGF-C overexpression increased lymphatic surface area in the tumor margin and lymphatic metastasis, these tumors contained no functional lymphatics, as assessed by four independent functional assays and immunohistochemical staining. These findings suggest that the functional lymphatics in the tumor margin alone are sufficient for lymphatic metastasis and should be targeted therapeutically.

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC) and mock-transfected cell lines (T241 fibrosarcoma and VEGFA embryonic stem cells) grown in murine dorsal skinfold chambers. VC tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

Lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 is thought to be restricted to lymph vessels and has been used as such to show that tumor lymphangiogenesis occurs on overexpression of lymphangiogenic factors in mouse tumor models. However, these studies have not yet been corroborated in human tumors. Here we show, first, that LYVE-1 is not exclusive to the lymph vessels. Indeed, LYVE-1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans. Surprisingly, LYVE-1 is absent from the angiogenic blood vessels of human liver tumors and only weakly present in the microcirculation of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. Second, we propose a novel approach to identify lymphatics in human and murine liver. By combining LYVE-1 and Prox 1 (a transcription factor) immunohistochemistry, we demonstrate that lymphatics are abundant in cirrhosis. In contrast, in human hepatocellular carcinoma and liver metastases, they are restricted to the tumor margin and surrounding liver. The absence of intratumor lymphatics in hepatocellular carcinomas and liver metastases may impair molecular and cellular transport in these tumors. Finally, the presence of LYVE-1 in liver sinusoidal endothelia suggests that LYVE-1 has functions beyond the lymph vascular system.

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.

Solid tumors produce both stimulators and inhibitors of angiogenesis. The suppression of metastases by some primary tumors has been attributed to the longer circulatory half-lives of the inhibitors. We propose that intrinsic differences in the physicochemical properties of these regulators may also explain focal suppression of angiogenesis within the primary tumor. We present a mathematical framework that describes production, diffusion, and degradation of these factors in tumor and host tissue and their effect on angiogenesis at local and distal sites. Results show focal suppression of angiogenesis, provide an explanation for tumor dormancy and focal necrosis, and predict a suppressive influence of primary tumors on angiogenesis at metastatic sites. They suggest generally that diffusible factors produced by tumors can stimulate responses in adjacent host tissue, preparing it for further tumor invasion. This study presents a new paradigm for the development of tumor necrosis and offers new insight into angiogenesis regulation and therapy. The framework established for modeling the competing effects of diffusible stimulators and inhibitors can be applied more generally to growth factors/inhibitors and other opposing factors produced in the tumor environment.