Tumor Regulation of Lymph Node Lymphatic Sinus Growth and Lymph Flow in Mice and in Humans.

The lymphatic vasculature collects and drains fluid and cells from the periphery through lymph nodes (LNs) for immune monitoring, and then returns lymph to the bloodstream. During immune responses LNs enlarge and remodel, featuring extensive growth of lymphatic sinuses (lymphangiogenesis). This LN lymphangiogenesis also arises in cancer, and is associated with altered lymph drainage through LNs. Studies of mouse solid tumor models identified lymphatic sinus growth throughout tumor-draining LNs (TDLNs), and increased lymph flow through the expanded sinuses. Mice developing B cell lymphomas also feature LN lymphangiogenesis and increased lymph flow, indicating that these changes occur in lymphoma as well as in solid tumors. These LN alterations may be key to promote tumor growth and metastasis to draining LNs and distant organs. Lymphatic sinus growth within the TDLN may suppress anti-tumor-immune responses, and/or the increased lymph drainage could promote metastasis to draining LNs and distant organs. Investigations of human cancers and lymphomas are now identifying TDLN lymphatic sinus growth and increased lymph flow, that correlate with metastasis and poor prognosis. Pathology assessment of TDLN lymphangiogenesis or noninvasive imaging of tumor lymph drainage thus could potentially be useful to assist with diagnosis and treatment decisions. Moreover, the expanded lymphatic sinuses and increased lymph flow could facilitate vaccine or drug delivery, to manipulate TDLN immune functioning or to treat metastases. The insights obtained thus far should encourage further investigation of the mechanisms and consequences of TDLN lymphatic sinus growth and lymph flow alterations in mouse cancer models, and in human cancer patients.

Tumor-induced alterations in lymph node lymph drainage identified by contrast-enhanced MRI.

BACKGROUND: To use high resolution MRI lymphography to characterize altered tumor-draining lymph node (TDLN) lymph drainage in response to growth of aggressive tumors. METHODS: Six mice bearing B16-F10 melanomas in one rear footpad were imaged by 3.0 Tesla (T) MRI before and after subcutaneous injection of Gadofosveset trisodium (Gd-FVT) contrast agent into both rear feet. Gd-FVT uptake into the left and right draining popliteal LNs was quantified and compared using Wilcoxon signed-rank test. Fluorescent dextran lymphography compared patterns of LN lymph drainage with the pattern of immunostained lymphatic sinuses by fluorescence microscopy. RESULTS: TDLNs exhibited greater Gd-FVT uptake than contralateral uninvolved LNs, although this difference did not reach significance (P < 0.06). Foci of contrast agent consistently surrounded the medulla and cortex of TDLNs, while Gd-FVT preferentially accumulated in the cortex of contralateral LNs at 5 and 15 min after injection. Fluorescent dextran lymphography confirmed these distinct contrast agent uptake patterns, which correlated with lymphatic sinus growth in TDLNs. CONCLUSION: 3.0T MRI lymphography using Gd-FVT identified several distinctive alterations in the uptake of contrast agent into TDLNs, which could be useful to identify the correct TDLN, and to characterize TDLN lymphatic sinus growth that may predict metastatic potential.

Tumors induce coordinate growth of artery, vein, and lymphatic vessel triads.

BACKGROUND: Tumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other. METHODS: The murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels. RESULTS: Each peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors. CONCLUSIONS: Implanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1.

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.

The actin-regulatory protein Hem-1 is essential for alveolar macrophage development.

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific actin-regulatory protein. Loss-of-function (LOF) variants in the NCKAP1L gene encoding Hem-1 have recently been found to result in primary immunodeficiency disease (PID) in humans, characterized by recurring respiratory infections, asthma, and high mortality. However, the mechanisms of how Hem-1 variants result in PID are not known. In this study, we generated constitutive and myeloid cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in lung immunity. We found that Hem-1-deficient mice accumulated excessive surfactant and cell debris in airways (pulmonary alveolar proteinosis) due to impaired development of alveolar macrophages (AMs) and reduced expression of the AM differentiation factor Pparg. Residual Hem-1-deficient AMs shifted to a proinflammatory phenotype, and Hem-1-deficient neutrophils and monocytes failed to migrate normally. Myeloid cell-specific Hem-1-deficient mice exhibited increased morbidity following influenza A virus or Streptococcus pneumoniae challenge. These results provide potential mechanisms for how LOF variants in Hem-1 result in recurring respiratory diseases.

Lymph node mapping in the mouse.

Accurate identification of lymph nodes in the mouse is critical for studies of tumor metastasis, and of regional immune responses following immunization. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques, so that larger rats have been used to characterize rodent lymphatic drainage. We developed techniques injecting dye into the mouse footpad or tail, to label the lymphatic drainage of the hind leg and flank, pelvic viscera, prostate and mammary glands. While lymphatic drainage patterns were similar in mice and rats, the inguinal lymph nodes showed distinct differences in afferent and efferent drainage. These techniques allow accurate and rapid identification of lymph nodes and lymphatic drainage in normal as well as diseased mice.

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses.

Lymphocyte- and leukocyte-mediated lymph node (LN) lymphatic sinus growth (lymphangiogenesis) is involved in immune responses and in diseases including cancer and arthritis. We previously discovered a 10.1.1 Ab that recognizes the lymphatic endothelial cell (LEC) surface protein mCLCA1, which is an interacting partner for LFA1 and Mac-1 that mediates lymphocyte adhesion to LECs. Here, we show that 10.1.1 Ab treatment specifically induces LEC proliferation, and influences migration and adhesion in vitro. Functional testing by injection of mice with 10.1.1 Ab but not control hamster Abs identified rapid induction of LN LEC proliferation and extensive lymphangiogenesis within 23 h. BrdU pulse-chase analysis demonstrated incorporation of proliferating LYVE-1-positive LEC into the growing medullary lymphatic sinuses. The 10.1.1 Ab-induced LN remodeling involved coordinate increases in LECs and also blood endothelial cells, fibroblastic reticular cells, and double negative stroma, as is observed during the LN response to inflammation. 10.1.1 Ab-induced lymphangiogenesis was restricted to LNs, as mCLCA1-expressing lymphatic vessels of the jejunum and dermis were unaffected by 23 h 10.1.1 Ab treatment. These findings demonstrate that 10.1.1 Ab rapidly and specifically induces proliferation and growth of LN lymphatic sinuses and stroma, suggesting a key role of mCLCA1 in coordinating LN remodeling during immune responses.

Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy.

Tumor-draining lymph nodes (TDLNs) often enlarge in human cancer patients and in murine tumor models, due to lymphocyte accumulation and lymphatic sinus growth. B lymphocytes within TDLNs can drive lymph node hypertrophy in response to tumor growth, however little is known about the mechanisms directing the preferential accumulation of B lymphocytes relative to T cells in enlarging TDLNs. To define why B and T lymphocytes accumulate in TDLNs, we quantified lymphocyte proliferation, apoptosis, entry, and exit in TDLNs versus contralateral non-TDLNs (NTDLNs) in a footpad B16-F10 melanoma mouse model. B and T lymphocyte proliferation and apoptosis were increased as the TDLNs enlarged, although relative rates were similar to those of NTDLNs. TDLN entry of B and T lymphocytes via high endothelial venules was also modestly increased in enlarged TDLNs. Strikingly, the egress of B cells was strongly reduced in TDLNs versus NTDLNs, while T cell egress was modestly decreased, indicating that regulation of lymphocyte exit from TDLNs is a major mechanism of preferential B lymphocyte accumulation. Surface sphingosine-1-phosphate receptor 1 (S1PR1) which binds S1P and signals lymphocyte egress, exhibited greater downregulation in B relative to T lymphocytes, consistent with preferential retention of B lymphocytes in TDLNs. TDLN lymphocytes did not activate surface CD69 expression, indicating a CD69-independent mechanism of downregulation of S1PR1. B and T cell trafficking via afferent lymphatics to enter TDLNs also increased, suggesting a pathway for accumulation of tumor-educated lymphocytes in TDLNs. These mechanisms regulating TDLN hypertrophy could provide new targets to manipulate lymphocyte responses to cancer.

Reduced Myc overexpression and normal B-cell differentiation mediate resistance to avian leukosis virus lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in tumor-susceptible chicken strains after proviral integration within the c-myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed follicles. Line 6(3) strain chickens are resistant to ALV tumorigenesis, largely failing to develop Myc-transformed follicles, although they show similar levels of ALV infection and integration as lymphoma-susceptible strains. Immunohistochemical analysis determined that the transformed follicles that do arise in lymphoma-resistant birds show much lower and more variable Myc overexpression than those of susceptible birds. This reduced Myc overexpression fails to block B-cell differentiation in resistant birds, while high Myc consistently blocks development at a late embryo stage in susceptible birds. This failure of Myc to block differentiation results in a normal pattern of posthatching bursal emigration in resistant transformed follicles, while transformed follicles of susceptible birds grow rapidly due to blocked emigration. Forced Myc overexpression produces transformed follicles in resistant birds, indicating that resistant lymphocytes can tolerate high Myc expression. The coding sequence and expression of the endogenous c-myc gene is the same in resistant and susceptible birds, suggesting that genetic resistance is instead mediated by reduced ALV LTR enhancer-driven transcription in the target lymphocytes of resistant birds.

Regulatory B cells preferentially accumulate in tumor-draining lymph nodes and promote tumor growth.

Our previous studies found that B16-F10 melanoma growth in the rear footpad of immunocompetent mice induces marked B cell accumulation within tumor-draining popliteal lymph nodes (TDLN). This B cell accumulation drives TDLN remodeling that precedes and promotes metastasis, indicating a tumor-promoting role for TDLN B cells. Here we show that phenotypic characterization of lymphocytes in mice bearing B16-F10 melanomas identifies preferential accumulation of T2-MZP B cells in the TDLN. Comparison of non-draining LNs and spleens of tumor-bearing mice with LNs and spleens from naïve mice determined that this pattern of B cell accumulation was restricted to the TDLN. B cell-deficient and immunocompetent mice reconstituted with T2-MZP B cells but not with other B cell subsets displayed accelerated tumor growth, demonstrating that T2-MZP B cells possess regulatory activity in tumor-bearing mice. Unlike splenic regulatory B cells, however, these TDLN B cells did not exhibit increased IL-10 production, nor did they promote Treg generation in the TDLN. These findings demonstrate that tumors initially signal via the lymphatic drainage to stimulate the preferential accumulation of T2-MZP regulatory B cells. This local response may be an early and critical step in generating an immunosuppressive environment to permit tumor growth and metastasis.

Culturing purifies murine lymph node lymphatic endothelium.

BACKGROUND: Lymph node (LN) lymphatic sinuses transport lymph, cells, and antigens from the periphery through the LN. The lymphatic endothelium lining these sinuses appears to be an important contributor to the lymph node immune response. It has been challenging to obtain sufficient LN lymphatic endothelial cells for investigation of their functions, as they are minor constituents of LNs. METHODS AND RESULTS: A procedure was developed to purify lymphatic endothelial cells (LEC) from murine LNs, which yields large numbers of primary LN LEC. Two-dimensional in vitro cultures of dissociated LN stromal cells initially consist of multiple cell types, and then rapidly evolve to produce pure cultures of lymphatic endothelium within a few passages. One million LEC can be harvested after 4 weeks of culture, and much larger cell numbers can be obtained by continued culturing over long periods. The LEC cultures maintain endothelial morphology and expression of LEC markers, and preserve the same slow growth characteristics over at least 20 passages. The LEC cultures readily form tubes in Matrigel at early and at late passages, resembling those formed by LEC lines. CONCLUSIONS: A simple and economical approach to obtain purified primary murine LN LEC was developed for in vitro studies of their function. The morphology, growth characteristics, and functional behavior of these cells in tube formation assays did not change between initial and long-term passages. Large numbers of these cells can be harvested after long-term passage, so that they can be studied in biochemical and biological assays.

Evaluation of reference genes for quantitative PCR analysis of mouse lymphocytes.

Normalization to a reference gene is the method of choice for quantitative PCR analysis. The stability of reference genes is critical for accurate gene expression analysis, as significant variations in reference gene expression can alter experimental results and conclusions. In this study, we evaluated the expression stability of five commonly used reference genes found in mouse lymphocytes. Using NormFinder and BestKeeper algorithms, we consistently show that ubiquitin C (Ubc) is the optimal reference gene for normalizing qPCR data obtained from mouse lymphocytes, whereas beta-actin (Actb) is not a suitable reference gene due to its extensive variability in expression. Our findings emphasize the importance of validating reference genes for qPCR analyses. We provide a shortlist of reference genes to use for normalization and recommend freely available software programs as a rapid approach to validate potential reference genes.

B lymphocytes promote lymphogenous metastasis of lymphoma and melanoma.

The prognosis of patients with many types of cancers correlates with the degree of metastasis to regional lymph nodes (LNs) and vital organs. However, the mechanisms and route of cancer cell metastasis are still unclear. Previous studies determined that B-cell accumulation in tumor-draining LNs (TDLNs) induces lymphatic sinus growth (lymphangiogenesis) and increases lymph flow, which could actively promote tumor dissemination through the lymphatic system. Using young Eµ-c-Myc mice that feature LN B-cell expansion as hosts for tumor transplants, we show that subcutaneously implanted lymphomas or melanomas preferentially spread to TDLNs over non-TDLNs, thus demonstrating that these tumors initially metastasize through lymphatic rather than through hematogenous routes. In addition, the rate and amount of tumor dissemination is greater in Eµ-c-Myc mice versus wild-type hosts, which correlates with LN B-cell accumulation and lymphangiogenesis in Eµ-c-Myc hosts. The increased lymphatic dissemination in Eµ-c-Myc hosts is further associated with rapid hematogenous tumor spread of subcutaneously implanted lymphomas, suggesting that TDLN metastasis secondarily drives lymphoma spread to distant organs. In contrast, after intravenous tumor cell injection, spleen metastasis of lymphoma cells or lung metastasis of melanoma cells is similar in Eµ-c-Myc and wild-type hosts. These studies demonstrate that the effect of Eµ-c-Myc hosts to promote metastasis is limited to the lymphatic route of dissemination. TDLN B-cell accumulation, in association with lymphangiogenesis and increased lymph flow, thus significantly contributes to dissemination of lymphomas and solid tumors, providing new targets for therapeutic intervention to block metastasis.

Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.

Peripheral immune tolerance is generally thought to result from cross-presentation of tissue-derived proteins by quiescent tissue-resident dendritic cells to self-reactive T cells that have escaped thymic negative selection, leading to anergy or deletion. Recently, we and others have implicated the lymph node (LN) stroma in mediating CD8 T cell peripheral tolerance. We demonstrate that LN-resident lymphatic endothelial cells express multiple peripheral tissue antigens (PTAs) independent of the autoimmune regulator (Aire). They directly present an epitope derived from one of these, the melanocyte-specific protein tyrosinase, to tyrosinase-specific CD8 T cells, leading to their deletion. We also show that other LN stromal subpopulations express distinct PTAs by mechanisms that vary in their Aire dependence. These results establish lymphatic endothelial cells, and potentially other LN-resident cells, as systemic mediators of peripheral immune tolerance.

Dynamic contrast-enhanced magnetic resonance imaging of tumor-induced lymph flow.

The growth of metastatic tumors in mice can result in markedly increased lymph flow through tumor-draining lymph nodes (LNs), which is associated with LN lymphangiogenesis. A dynamic magnetic resonance imaging (MRI) assay was developed, which uses low-molecular weight gadolinium contrast agent to label the lymphatic drainage, to visualize and quantify tumor-draining lymph flow in vivo in mice bearing metastatic melanomas. Tumor-bearing mice showed greatly increased lymph flow into and through draining LNs and into the bloodstream. Quantitative analysis established that both the amount and the rate of lymph flow through draining LNs are significantly increased in melanoma-bearing mice. In addition, the rate of appearance of contrast media in the bloodstream was significantly increased in mice bearing melanomas. These results indicate that gadolinium-based contrast-enhanced MRI provides a noninvasive assay for high-resolution spatial identification and mapping of lymphatic drainage and for dynamic measurement of changes in lymph flow associated with cancer or lymphatic dysfunction in mice. Low-molecular weight gadolinium contrast is already used for 1.5-T MRI scanning in humans, which should facilitate translation of this imaging assay.

Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis.

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.

Abrupt decline in the rate of accumulation of total protein and yolk in postvitellogenic egg chambers ofDrosophila.

The accumulation of endogenously synthesized non-yolk proteins, and of exogenously derived yolk, was quantitated during oogenesis and embryogenesis ofDrosophila. Rates of non-yolk protein accumulation were calculated, and were correlated with polysome content at each developmental stage. Three distinct phases of non-yolk protein accumulation were observed: 1) relatively slow accumulation, lasting to stage 9 of oogenesis; 2) very rapid accumulation between stages 10 and 12 of oogenesis, when half of the protein of the mature egg is accumulated in less than 4 h; and 3) no further protein accumulation from stage 12 of oogenesis through at least the gastrula stage of embryogenesis. During phases 1 and 2, rates of non-yolk protein accumulation correlate well with the polysome content of egg chambers. Surprisingly, during the entire phase 3 the content of polysomes remains at high levels, even though no detectable protein accumulation occurs. This finding is in agreement with the low levels of protein synthesis that have been measured during early embryogenesis, and strongly suggests that late in oogenesis the efficiency of translation suddenly drops by about 20-fold. Moreover, our results imply that polysome content cannot always be directly correlated with protein synthetic activity.

B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis.

Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (E micro -c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) E micro -c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in E micro -c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from E micro -c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively.