Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognosis and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in human cancer remains to be determined.

Lymph nodes are initial sites for cancer metastasis in many solid tumors. However, their role in cancer progression is still not completely understood. Emerging evidence suggests that the lymph node microenvironment provides hospitable soil for the seeding and proliferation of cancer cells. Resident immune and stromal cells in the lymph node express and secrete molecules that may facilitate the survival of cancer cells in this organ. More comprehensive studies are warranted to fully understand the importance of the lymph node in tumor progression. Here, we will review the current knowledge of the role of the lymph node microenvironment in metastatic progression.

BackgroundInadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER.Principal FindingsMicroarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-Time PCR analyses confirmed that four of these factors, VEGFA, FGF2, angiogenin and IL8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGFA regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGFA promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGFA expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGFA gene, although its contribution to VEGFA transcription appeared to be fairly modest. We also found that VEGFA mRNA stability is increased in response to UPR activation, via activation of AMP kinase, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGFA protein is secreted at levels as high as or higher than that achieved in response to hypoxia.Conclusions and SignificanceOur results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGFA expression at transcriptional, post-transcriptional and post-translational levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer.

Background:The UPR and HIF signaling pathways are cytoprotective responses activated by inadequate cellular environments. Results: These pathways can cooperate to increase the expression of shared targets like VEGF. Conclusion:The UPR enhances HIF-1 transcriptional activity. Significance: The simultaneous activation of both pathways in tumors may result in greater vascularization.

Cancer patients with lymph node (LN) metastases have a worse prognosis than those without nodal disease. However, why LN metastases correlate with reduced patient survival is poorly understood. Recent findings provide insight into mechanisms underlying tumor growth in LNs. Tumor cells and their secreted molecules engage stromal, myeloid, and lymphoid cells within primary tumors and in the lymphatic system, decreasing antitumor immunity and promoting tumor growth. Understanding the mechanisms of cancer survival and growth in LNs is key to designing effective therapy for the eradication of LN metastases. In addition, uncovering the implications of LN metastasis for systemic tumor burden will inform treatment decisions. In this review, we discuss the current knowledge of the seeding, growth, and further dissemination of LN metastases.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema—fluid accumulation in tissue caused by impaired lymphatic vessel function—is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed both an acute reduction in lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation of MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)-controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.

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Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.