Tumor imaging with multicolor fluorescent protein expression

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics.     

Real-time imaging of lymphogenic metastasis in orthotopic human breast cancer

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.     

Lymphatic metastasis

Summary Lymphatic metastasis is an important mechanism in the spread of human cancer. During its course, tumor cells first penetrate the basement of membrane of the epithelium, in which they arise, and then the underlying connective tissue, carried partly by hydrostatic pressure. They enter the lymphatic partly by active movement, pass up the lymphatic trunk; they then settle and proliferate in the subcapsular sinus, penetrate its endothelium and proliferate and destroy the node. There are varied forms of immune response in the node and in human nodes often a complex fibrous and vascular response. The degree of lymphocytic response may be important for prognosis. The nodal reaction may be stimulated by release of antigens from the tumor.One of the most studied animal models of lymphatic metastasis is that which occurs in the politeal node after injection of tumor into the footpad. This model has been used to show that tumor cells enter lymphatics through gaps in endothelium, probably between endothelial cells, and that lymph nodes can destroy small numbers of tumor cells. Local immunotherapy and chemotherapy can sterilize a lymph node of tumor cells; the modes of treatment used have included intralymphatic injection and encapsulation of chemotherapeutic agents in liposomes. Prior radiotherapy may accelerate metastasis possibly by making tumor cells shed into lymphatic vessels. Lymph nodes are rather poor barriers to tumor cells. The prognostic significance of lymph node metastasis varies within tumor type; if hematogenous metastasis is early, then the presecce of lymph node metastasis is of lesser prognostic significance. Lymph nodes can probably destroy only small numbers of tumor cells.Tumor cell heterogeneity is of importance in many aspects of metastasis; while clonal variation may be of importance in determining lymph node metastasis, it is not yet clear how important this is, nor whether specific clones metastasize specifically to lymph nodes.Lymphography is well established in diagnosis of lymphatic metastasis. A recent interesting development has been to inject antibodies labeled with a radioactive label, and image the label in lymph nodes with a gamma-camera. If anti-tumor antibodies are used in this way it may be possible to detect lymph node metastasis.Within the expanding field of tumor metastasis, lymphatic metastasis needs much more attention, particularly in relation to the diagnosis and treatment of the lymphatic spread of human cancer.     

Imaging steps of lymphatic metastasis reveals that vascular endothelial growth factor-C increases metastasis by increasing delivery of cancer cells to lymph nodes: therapeutic implications

Preclinical and clinical studies positively correlate the expression of vascular endothelial growth factor (VEGF)-C in tumors and the incidence of lymph node metastases. However, how VEGF-C regulates individual steps in the transport of tumor cells from the primary tumor to the draining lymph nodes is poorly understood. Here, we image and quantify these steps in tumors growing in the tip of the mouse ear using intravital microscopy of the draining lymphatic vessels and lymph node, which receives spontaneously shed tumor cells. We show that VEGF-C overexpression in cancer cells induces hyperplasia in peritumor lymphatic vessels and increases the volumetric flow rate in lymphatics at the base of the ear by 40%. The increases in lymph flow rate and peritumor lymphatic surface area enhance the rate of tumor cell delivery to lymph nodes, leading to a 200-fold increase in cancer cell accumulation in the lymph node and a 4-fold increase in lymph node metastasis. In our model, VEGF-C overexpression does not confer any survival or growth advantage on cancer cells. We also show that an anti-VEGF receptor (VEGFR)-3 antibody reduces both lymphatic hyperplasia and the delivery of tumor cells to the draining lymph node, leading to a reduction in lymph node metastasis. However, this treatment is unable to prevent the growth of tumor cells already seeded in lymph nodes. Collectively, our results indicate that VEGF-C facilitates lymphatic metastasis by increasing the delivery of cancer cells to lymph nodes and therapies directed against VEGF-C/VEGFR-3 signaling target the initial steps of lymphatic metastasis.     

Evaluation of lymphatic flow pattern using indocyanine green fluorescence imaging in a highly metastatic mouse model

Recently, the feasibility of real‐time indocyanine green (ICG) fluorescence imaging–guided complete mesocolic excision in colon cancer surgery has been demonstrated; however, its application to the evaluation of lymphatic flow in widespread lymph node metastasis is uncertain. This study aimed to evaluate lymphatic flow using the real‐time ICG fluorescence imaging. A mouse model of subcutaneous inoculation of BJMC3879Luc2 cells, which have been demonstrated to highly metastasize to the lymph nodes, was used as an evaluation model. Tumor growth and lymphatic flow were monitored weekly by bioluminescent imaging and near‐infrared (NIR) fluorescence imaging, respectively. After sacrificing the mice, lymph node metastases were evaluated by bioluminescent imaging and histopathology. Lymphatic flows in a model of high lymph node metastasis were evaluated using NIR fluorescence imaging. Pathological metastases of bilateral axillary, femoral, and para‐aortic lymph nodes were detected in all inoculated mice (100%: 5/5). Real‐time NIR fluorescence imaging showed the primary lymphatic vessels staining through the metastatic lymph nodes as before the inoculation of the cancer cells. Hitherto, it has been considered that lymphatic flow was changed using the bypass pathway due to occlusion of the primary lymphatic vessels. In this presented study, real‐time ICG fluorescence imaging showed no changes in lymphatic flow after lymph node metastasis. Our results suggest that real‐time ICG fluorescence imaging may have potential for the guidance of colon cancer surgery in cases of widespread lymph node metastasis.     

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.     

Lymph node metastasis in breast cancer xenografts is associated with increased regions of extravascular drain, lymphatic vessel area, and invasive phenotype

Interactions between the tumor stromal compartment and cancer cells play an important role in the spread of cancer. In this study, we have used noninvasive in vivo magnetic resonance imaging (MRI) of two human breast cancer models with significantly different invasiveness, to quantify and understand the role of interstitial fluid transport, lymphatic-convective drain, and vascularization in the regional spread of breast cancer to the axillary lymph nodes. Quantitative fluorescence microscopy was done to morphometrically characterize lymphatic vessels in these tumors. Significant differences in vascular and extravascular transport variables as well as in lymphatic vessel morphology were detected between the two breast cancer models, which also exhibited significant differences in lymph node and lung metastasis. These data are consistent with a role of lymphatic drain in lymph node metastasis and suggest that increased lymph node metastasis may occur due to a combination of increased invasiveness, and reduced extracellular matrix integrity allowing increased pathways of least resistance for the transport of extravascular fluid, as well as tumor cells. It is also possible that lymph node metastasis occurred via the cancer cell-bearing tumoral lymphatic vessels. The congestion of these tumoral lymphatics with cancer cells may have restricted the entry and transport of macromolecules.     

Dynamics of lymphatic regeneration and flow patterns after lymph node dissection

Knowledge about the mechanisms of regeneration of the lymphatic vasculature after surgical trauma is essential for the development of strategies for the prevention and therapy of lymphedema. However, little is known about the alterations of lymphatic flow directly after surgical trauma. We investigated lymphatic function in mice using near-infrared imaging for a period of 4 weeks after surgeries that mimic sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), by removal of the popliteal lymph node (LN) alone or together with the popliteal fat pad, respectively. SLNB-like surgery did not cause changes in lymphatic drainage in the majority of cases. In contrast, lymphatic drainage impairment shown by collecting vessel rupture, dermal backflow and rerouting of lymph flow via collateral vessels were observed after ALND-like surgery. All collateral vessels drained to the inguinal LN. These results indicate that less invasive surgery prevents lymphatic decompensation. They also reveal the development and maturation of collateral lymphatic vessels after extensive surgical trauma, which reroute the flow of lymph towards a different LN. These findings might be helpful for the development of strategies to prevent and/or treat post-surgical lymphedema.     

Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development.     

Micro-magnetic resonance lymphangiography in mice using a novel dendrimer-based magnetic resonance imaging contrast agent

Major advances in cancer biology and immunology have been gained using mouse models. However, very few methods are currently available to visualize the deep lymphatic system. A new micro-magnetic resonance lymphangiography (MRL) method in mice, which uses dendrimer-based magnetic resonance imaging contrast agents, was developed. Micro-MRL imaging clearly visualized most of the mouse lymphatic system, including both lymphatics and lymph nodes. This method could detect and distinguish among dilation of lymphatic vessels in a lymphangitis model, proliferative or neoplastic lymph node swellings in a lymphoproliferative model, and inflammatory lymph node swellings in an infection/inflammation model. Changes in the lymphoid system of transgenic mice overexpressing interleukin-15 could be visualized. Abnormal enlarged lymph nodes identified by micro-MRL were selectively removed and analyzed to demonstrate their cell type, receptor expression, and clonality in individual mice. We conclude that the enhanced resolution of this noninvasive micro-MRL can detect and classify lymphatic and lymph node abnormalities in mice, which should have wide applicability to the study of immunology and cancer in both experimental animals and clinical medicine.     

Lymphatic mapping and lymphatic endothelial cell isolation in colorectal cancer patients

Background: Sentinel lymph node (SLN) biopsy has already been established as a common procedure with malignant lymphoma and breast cancer. In colorectal cancer, however, the application of the SLN theory remains uncommon and its clinical significance is also unclear. In addition, the characteristics of the lymphatic vessels that connect SLNs or the lymphatic endothelial cells have been unclear. Our purpose is to determine the feasibility and accuracy of SLN mapping by intraoperative subserosal dye injection and to develop a novel method for the isolation of anatomically defined lymphatic endothelial cells. Methods: SLN biopsy by the subserous dye injection method (patent blue) was conducted in 36 patients with colorectal cancer for which curative resection was possible (stage 0: two cases, stage I: 18 cases, stage II: four cases, stage III: 12 cases), with additional systematic lymph node dissection. Endothelial cells were isolated from lymphatic vessels identified at the time of the SLN biopsy by the collagenase II perfusion method, and we tried to transfer them into a culture system with an endothelial cell‐specific medium. Results: SLNs could be identified in 34 cases, providing a 94.4% detection rate. The total number of resected lymph nodes was 705 and 72 of those nodes were confirmed as SLNs (10.2%). Ten metastasis‐positive nodes were found in SLNs (13.9%), and the mean number of identified SLNs per case was 2.0. No complications or toxicity associated with the dye injection were observed. In addition, cells isolated from the lymphatic vessels removed formed some colonies and the expression of lymphatic endothelial cell‐specific VEGFR‐3 was observed. Conclusion: SLNs mapping using subserosal dye injection is a feasible technique that would make the range of lymph node dissection appropriate for colorectal cancer, which is applicable to cases with advanced cancer. This new method was established to isolate lymphatic endothelial cells from resected lymphatic vessels and to culture them, which may contribute to further study of the molecular mechanisms of lymphangiogenesis.     

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE‐1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE‐1, and investigated the roles of LYVE‐1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)‐fused mouse LYVE‐1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP‐fused mouse LYVE‐1 proteins in a GFP expression‐dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE‐1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA‐MB‐231 xenograft models. This shows that LYVE‐1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.     

Mannose receptor (MR) and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium

Although approximately 50% of cancers give rise to metastases via the lymphatic system, the mechanisms mediating this process have remained unknown. In this study, we have investigated the role of two lymphatic endothelial molecules, the mannose receptor (MR) and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 in adhesion of malignant cells to the lymphatic endothelium, and analyzed their expression in two clinical series consisting of squamous cell cancers of the head and neck (n = 17) and breast cancers (n = 72). Affinity of the tested head and neck cancer cell lines to the lymphatic endothelium varied greatly, but adhesion of all cell lines was dependent on both the MR and CLEVER-1. Almost all cancer specimens contained peritumoral vessels that expressed CLEVER-1 and MR, and also the intratumoral lymph vessels often expressed them in both tumor types. However, only intratumoral expression of these molecules seems to be essential for metastatic spread to the regional lymph nodes. Only 8 (22%) of the 36 axillary node-negative breast carcinomas expressed the MR on the intratumoral lymph vessels as compared with 16 (50%) of the 32 node-positive carcinomas (P = 0.017), and all eight head and neck carcinoma patients with regional lymph node metastases at diagnosis had tumors that expressed CLEVER-1 on the intratumoral lymph vessels. These data suggest a role for both the MR and CLEVER-1 in directing the traffic of cancer cells within the lymphatic system.     

Color-coded real-time subcellular fluorescence imaging of the interaction between cancer and host cells in live mice

Stromal cells are essential for tumor growth. Stromal cells interact with cancer cells during tumor growth and progression. We report here the development of a tri-color imageable mouse model to visualize the interaction between host cells and cancer cells. To observe subcellular cancer cell dynamics in vivo, HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-green fluorescent protein (GFP) and with retroviral red fluorescent protein (RFP) in the cytoplasm. HT-1080-GFP-RFP cells were sprinkled over a skin-flap in transgenic GFP immunocompetent mice. After 24 h, the mice were imaged with an Olympus IV100 laser scanning microscope. HT-1080-GFP-RFP cells were visualized surrounded by host-derived lymphocytes and macrophages both expressing GFP. It was possible to observe host GFP macrophages contacting, engulfing, and digesting dual-color HT-1080-GFP-RFP cells in real time. The dual-color cancer cells were readily visible after being engulfed in the GFP macrophages. Other cancer cells were visualized being killed by lymphocytes. The results of this study show that differentially labeling cells with spectrally-distinct fluorescent protein can allow subcellular-resolution imaging of cell-cell interactions between host and cancer cells.     

Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer.

Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Vascular endothelial growth factor C (VEGF-C) is known to be the only growth factor for the lymphatic vascular system, and its receptor has been identified as Flt4. To clarify the mechanism of lymphatic invasion in cancer, we studied the expression of VEGF-C and flt4 genes in gastric cancer tissues. VEGF-C mRNA was mainly expressed in primary tumors (15 of 32; 47%), but the frequency of VEGF-C mRNA expression was low in normal mucosa (4 of 32; 13%). In primary tumors, there was a significant relationship between VEGF-C and flt4 mRNA expression. In contrast, Flt4 was mainly expressed on the lymphatic endothelial cells but not in cancer cells. A strong correlation was found between VEGF-C expression and lymph node status, lymphatic invasion, venous invasion, and tumor infiltrating patterns. Cancer cells in the lymphatic vessels frequently showed intracytoplasmic VEGF-C immunoreactivity. Furthermore, there was a close correlation between VEGF-C tissue status and the grade of lymph node metastasis. Patients with high expression of VEGF-C protein had a significantly poorer prognosis than did those in low VEGF-C expression group. By the Cox regression model, depth of wall invasion, lymph node metastasis, and VEGF-C tissue status emerged as independent prognostic parameters, and the VEGF-C tissue status was ranked third as an independent risk factor for death. These results strongly suggest that cancer cells producing VEGF-C may induce the proliferation and dilation of lymphatic vessels, resulting in the development of invasion of cancer cells into the lymphatic vessel and lymph node metastasis.     

CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor‐mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7‐dependent signaling in MDA‐MB‐231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7‐dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA‐MB‐231–derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE‐1⁺ intratumoral and peritumoral lymphatic vessels. In the three‐dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21‐reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand–expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.     

乳腺癌淋巴管生成与肿瘤转移的关系

目的：探讨乳腺癌肿瘤淋巴管生成与肿瘤转移的关系。方法：免疫组织化学SP法进行VEGFR-3染色标记89例原发性乳腺癌肿瘤淋巴管。结果：所有病例均有不同程度淋巴管生成，但以肿瘤间质组织中淋巴管生成为主，癌巢中未见明显的成形淋巴管。肿瘤淋巴管密度与乳腺癌临床分期和腋淋巴结转移呈正相关，临床分期越晚，肿瘤淋巴管密度越高（P〈0．05）；腋淋巴结转移组的肿瘤淋巴管密度比无淋巴结转移组高（P〈0．05）。结论：乳腺癌淋巴管密度与腋淋巴结转移呈正相关．淋巴管生成主要发生在肿瘤间质组织中。     

Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer.

PURPOSE: The molecular mechanisms underlying lymph node metastasis are poorly understood, despite the well-established clinical importance of lymph node status in many human cancers. Recently, vascular endothelial growth factor (VEGF)-C and VEGF-D have been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion via activation of VEGF receptor-3. The purpose of this study was to determine the expression pattern of the VEGF-C/VEGF-D/VEGF receptor-3 axis in prostate cancer and its relationship with lymph node metastasis. EXPERIMENTAL DESIGN: The expression pattern of VEGF-C, VEGF-D, and VEGF receptor-3 in localized prostate cancer specimens (n = 37) was determined using immunohistochemistry. RESULTS: Widespread, heterogeneous staining for VEGF-C and VEGF-D was observed in all cancer specimens. Intensity of VEGF-C staining was lower in benign prostate epithelium than in adjacent carcinoma, whereas no difference between benign epithelium and carcinoma was observed for VEGF-D staining. VEGF receptor-3 immunostaining was detected in endothelial cells of lymphatic vessels in 18 of 37 tissue samples. The presence of VEGF receptor-3-positive vessels was associated with lymph node metastasis (P = 0.0002), Gleason grade (P < 0.0001), extracapsular extension (P = 0.0382), and surgical margin status (P = 0.0069). In addition, VEGF receptor-3 staining highlighted lymphatic invasion by VEGF-C-positive/VEGF-D-positive carcinoma cells. CONCLUSIONS: Together, these results suggest that paracrine activation of lymphatic endothelial cell VEGF receptor-3 by VEGF-C and/or VEGF-D may be involved in lymphatic metastasis. Thus the VEGF-C/VEGF-D/VEGF receptor-3 signaling pathway may provide a target for antilymphangiogenic therapy in prostate cancer.