Alanna Ruddell

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.

Angiogenesis is an early event in the generation of myc -induced lymphomas

Angiogenesis was identified as an early consequence of myc gene overexpression in two models of retroviral lymphomagenesis. Avian leukosis virus (ALV) induces bursal lymphoma in chickens after proviral c-myc gene integration, while the HB-1 retrovirus carries a v-myc oncogene and also induces metastatic lymphoma. Immunohistochemical studies of the effects of increased c-myc or v-myc overexpression revealed early angiogenesis within myc-transformed bursal follicles, which persisted in lymphomas and metastases. Abnormal vessel growth was consistently detected within 13 days after transplantation of a few myc-overexpressing progenitors into ablated bursal follicles, suggesting that these angiogenic changes may support the initial expansion of tumor precursors, as well as later stage lymphomagenesis. Conditioned media from myc-overexpressing B cell lines promoted proliferation of vascular endothelium in vitro, while media from B cells expressing low myc levels showed little effect. Moreover, ectopic myc overexpression in the low myc B cell lines increased production of the endothelial growth activity, indicating that myc induces secretion of angiogenic factors from B cells. These findings demonstrate that myc overexpression in lymphocytes generates an angiogenic phenotype in vitro as well as in vivo.

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 Burkitts 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.

p19/Arf and p53 suppress sentinel lymph node lymphangiogenesis and carcinoma metastasis

The ability of tumor cells to metastasize is increasingly viewed as an interaction between the primary tumor and host tissues. Deletion of the p19/Arf or p53 tumor suppressor genes accelerates malignant progression and metastatic spread of 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced squamous cell carcinomas, providing a model system to address mechanisms of metastasis. Here, we show that benign pre-metastatic papillomas from wild-type mice trigger lymphangiogenesis within draining lymph nodes, whereas there is no growth of primary tumor lymphatic vessels. Lymph node lymphangiogenesis is greatly accelerated in papilloma-bearing p19/Arf- or p53-deficient mice, which coincides with the greater propensity of these tumors to progress to carcinomas and to metastasize. The extent of accumulation of B cells within the tumor-draining lymph nodes of wild-type mice predicted the level of lymph node lymphangiogenesis and metastatic potential. Arf or p53 deficiency strongly accelerated lymph node immune cell accumulation, in a manner that was associated with the extent of lymph node lymphatic sinus growth. This immune cell accumulation and lymph node lymphangiogenesis phenotype identifies host anti-tumor responses that could drive metastatic spread of cancers via the lymphatics.

Tissue-specific lability and expression of avian leukosis virus long terminal repeat enhancer-binding proteins.

Avian leukosis virus (ALV) induces bursal lymphomas in chickens, after proviral integration next to the cellular myc proto-oncogene, and subsequent c-myc hyperexpression. Our previous work suggested that labile or short-lived cellular proteins interact with the viral long terminal repeat (LTR) enhancer, and binding of these proteins appeared to be essential for high rates of LTR-enhanced transcription (A. Ruddell, M. Linial, W. Schubach, and M. Groudine, J. Virol. 62:2728-2735, 1988). This lability is specific for B-lymphoid cell types, since T cells and fibroblasts show stable high rates of LTR-enhanced transcription and stable LTR-binding activity. Moreover, the lability of these proteins may be important in determining susceptibility to bursal lymphoma. In this study, we separated and characterized the labile and stable LTR-binding proteins and examined their lability and expression in different cell types. Gel shift and DNase I footprinting analyses indicated that at least five proteins interact with the 140-base-pair LTR enhancer region. These proteins were distinct by several criteria, including lability or stability after inhibition of protein synthesis, resistance to heat denaturation, chromatographic behavior, and expression in different cell types. Two binding proteins were present in many cell types and were specifically labile in B cells. A third binding protein showed hematopoietic-cell-type-specific expression and was also labile in B cells. These findings indicate that there is tissue-specific modulation of the lability and expression of ALV LTR-binding proteins, which may be important for regulation of LTR transcription enhancement and ALV bursal lymphomagenesis.

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.

Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.

Avian leukosis virus induces lymphoma in chickens after proviral integration within the c-Myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed bursal follicles. The clonal expansion of these follicles allowed us to examine how Myc influences cell differentiation, growth, and apoptosis in lymphoid progenitors soon after the onset of Myc overexpression. Immunohistochemical analysis of developmental markers established that Myc overexpression consistently blocks lymphocyte differentiation at a late embryonic stage. Myc-transformed follicles also grow much more rapidly than normal follicles. This rapid growth is not mediated by suppression of apoptosis, as normal and Myc-transformed follicles showed similar rates of cell death by TUNEL immunohistochemical analyis of cells undergoing DNA degradation. Measurements of DNA synthesis and mitotic index showed modest effects of Myc to increase lymphocyte proliferation, as normal lymphocytes already divide rapidly. The major mechanism mediating rapid growth of transformed follicles instead involved failure of myc-overexpressing lymphocytes to emigrate from transformed follicles, while normal lymphocytes actively emigrate after hatching, as measured by BrdU pulse-chase labeling and immunohistochemical measurements. This failure to undergo the normal program of differentiation and subsequent bursal retention of lymphocytes accounts for most of the growth of transformed follicles, while Myc-induced proliferation makes a smaller contribution.

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 63 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.

Tumor-induced lymph node alterations detected by MRI lymphography using gadolinium nanoparticles

Contrast-enhanced MRI lymphography shows potential to identify alterations in lymph drainage through lymph nodes (LNs) in cancer and other diseases. MRI studies have typically used low molecular weight gadolinium contrast agents, however larger gadolinium-loaded nanoparticles possess characteristics that could improve the specificity and sensitivity of lymphography. The performance of three gadolinium contrast agents with different sizes and properties was compared by 3T MRI after subcutaneous injection. Mice bearing B16-F10 melanoma footpad tumors were imaged to assess tumor-induced alterations in lymph drainage through tumor-draining popliteal and inguinal LNs versus contralateral uninvolved drainage. Gadolinium lipid nanoparticles were able to identify tumor-induced alterations in contrast agent drainage into the popliteal LN, while lower molecular weight or albumin-binding gadolinium agents were less effective. All of the contrast agents distributed in foci around the cortex and medulla of tumor-draining popliteal LNs, while they were restricted to the cortex of non-draining LNs. Surprisingly, second-tier tumor-draining inguinal LNs exhibited reduced uptake, indicating that tumors can also divert LN drainage. These characteristics of tumor-induced lymph drainage could be useful for diagnosis of LN pathology in cancer and other diseases. The preferential uptake of nanoparticle contrasts into tumor-draining LNs could also allow selective targeting of therapies to tumor-draining LNs.

Resistance to avian leukosis virus lymphomagenesis occurs subsequent to proviral c- myc integration

Most chicken strains are highly susceptible to avian leukosis virus (ALV) induction of bursal lymphoma, involving proviral integration within the c-myc proto-oncogene, while certain strains are genetically resistant to lymphomagenesis. A nested PCR assay was developed to analyse the appearance of proviral c-myc integrations after ALV infection of lymphoma-susceptible birds, and to determine whether these integrations arise in lymphoma-resistant birds. Proviral c-myc integrations are detected in bursa and other tissues from 6 day-old lymphoma-susceptible birds infected as embryos. The abundance of bursal cells carrying these integrations increases roughly 40-fold by 35 days of age, indicating that these cells hyperproliferate within the bursal environment. Bursal cells with proviral c-myc integrations also arise soon after infection of lymphoma-resistant embryos. However, these cells expand much more slowly than cells from lymphoma-susceptible birds. Both strains show the same rate of viral infection, so that resistance to lymphomagenesis occurs at a step subsequent to proviral c-myc integration. Proviral c-erbB gene integrations arise at the same frequency in bursa and other tissues of both strains, and they do not increase in abundance during development. These findings indicate that the mechanism of resistance to lymphomagenesis involves specific inhibition of cells with proviral c-myc integrations within the bursa.