Background. In accord with the cancer stem cell (CSC) theory, only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from head and neck squamous cell cancer (HNSCC). Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs.Methods. 1 Despite advances in therapy that have improved quality of life, survival rates have remained static for many years. Mortality from this disease remains high because of the development of distant metastasis and the emergence of treatment-resistant local and regional recurrences. To develop more effective therapies for head and neck squamous cell cancer (HNSCC), it is essential that we gain a deeper understanding of the biology Correspondence to: M. E. Prince

Objective Subpopulations of highly tumorigenic cells, which have the unique capacity to self-renew and produce differentiated progeny, have been identified in multiple malignancies. In head and neck squamous cell carcinoma (HNSCC), this subpopulation of cells, termed cancer stem cells (CSCs) are contained within the population with high CD44 expression. It has been postulated that CSCs play a role in invasion and metastasis; however, there is little evidence to support this theory. We designed in vitro and in vivo models of metastasis to study the behavior of CSCs in HNSCC. Design Cells were sorted for CD44 expression using flow cytometry. Sorted cells were used in an in vitro invasion assay. For in vivo studies, CSCs and non-CSCs were injected into the tail veins of mice, and lungs were either harvested or imaged to evaluate for metastases. Results In vitro, CD44high cells were more motile but less invasive than CD44low cells. In vivo, 4/5 mice injected with CD44high cells and 0/5 mice injected with CD44low cells formed lung metastases. Two of the metastases arose from CSCs from a primary tumor and three from CSCs from HNSCC cell lines. Conclusions In vitro, CSCs do not have an increased ability to invade through basement membrane, but they do migrate more efficiently through a porous barrier. In contrast, CSCs formed metastases quite efficiently in vivo, whereas non-CSCs did not form metastases at all. This phenomenon could be due to enhanced migratory capacity of CSCs, which may be more important than basement membrane degradation in vivo.

BACKGROUND Few human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established UM-SCC-104, a new HPV(+) HNSCC cell linefrom a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSC). METHODS Tumor cells were tested for biomarker expression by immunohistology and the presence of HPV was assessed by several methods. RESULTS UM-SCC-104 has a unique genotype, contains HPV-16 and expresses E6/E7. Inoculation of (Aldehyde Dehydrogenase) ALDH(+) and ALDH(−) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, while ALDH(−) cells did not produce tumors. CONCLUSIONS UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of EGFR and a functionally significant proportion of ALDH(+) CSC.

With recent advances in molecular techniques, collecting blood from birds has become a common practice among field ornithologists. There are a variety of techniques for collecting blood samples and numerous caveats for how samples should be processed, depending on the research question being asked. Currently, few resources are available for individuals learning how to collect blood from birds or needing more information about how to process blood samples. Here, I describe commonly used methods for collecting, processing, and storing blood for particular research objectives, and provide answers to frequently asked questions about blood collection. The information provided is intended primarily for investigators working with passerines, but many techniques and suggestions are applicable to other avian taxa.

Whereas migrating birds have been implicated in the spread of West Nile virus (WNV), there is no direct evidence of birds actively migrating while infectious. The role of birds in WNV dispersal is difficult to assess in the field. However, this role can be evaluated experimentally because birds in migratory disposition display increased locomotor activity or restlessness under captive conditions. We tested the following hypotheses: (1) migrating passerine birds continue to exhibit migratory activity while infectious with WNV and(2) the migratory state of the individual affects the magnitude of viremia. We examined the migratory activity of two neoarctic-neotropical passerine migrants, SwainsonÕs thrush (Catharus ustulatus) and gray catbird (Dumetella carolinensis), during acute WNV infection. All gray catbirds and six of nine SwainsonÕs thrushes exhibited migratory activity while infectious. Moreover, migratory status did not appear to influence viremia titers, as might be expected if individuals were immunosuppressed during migration. Therefore, we demonstrate that migrating passerine birds are potential dispersal vehicles for WNV.

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Keywords: antitumor immunity, cancer stem cell, dendritic cell, metastasis, vaccine Abbreviations: ALDH, aldehyde dehydrogenase; CSC, cancer stem cell; CSC-DC, CSC lysate-pulsed dendritic cell; DC, dendritic cell; H-DC, heterogeneous, unsorted tumor cell lysate-pulsed dendritic cell; RT, radiation therapy; qRT-PCR, real time quantitative PCR.The inability to target cancer stem cells (CSC) may be a significant factor contributing to treatment failure. We have developed a strategy to target the CSC populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DCs). The CSC-DC vaccine was administered in the adjuvant setting after localized radiation therapy of established tumors. Using mouse models we demonstrated that DCs pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (termed CSC-DC) significantly inhibited tumor growth, reduced development of pulmonary metastases and prolonged survival. The effect was associated with downregulation of chemokine (C-C motif) receptors CCR7 and CCR10 in tumor cells and decreased expression of the chemokine (C-C motif) ligands CCL21, CCL27 and CCL28 in lung tissue. The CSC-DC vaccine significantly reduced ALDH high CSC frequency in primary tumors. Direct targeting of CSCs was demonstrated by the specific binding of IgG produced by ALDH high CSC-DC vaccineprimed B cells to ALDH high CSCs, resulting in lysis of these target CSCs in the presence of complement. These data suggest that the CSC-DC vaccine approach may be useful in the adjuvant setting where local and systemic relapse are high after conventional treatment of cancers.

Migrating waterfowl are implicated in the global spread of influenza A viruses (IAVs), and mallards (Anas platyrhynchos) are considered a particularly important IAV reservoir. Prevalence of IAV infection in waterfowl peaks during autumn pre-migration staging and then declines as birds reach wintering areas. Migration is energetically costly and birds often experience declines in body condition that may suppress immune function. We assessed how body condition affects susceptibility to infection, viral shedding and antibody production in wild-caught and captive-bred juvenile mallards challenged with low pathogenic avian influenza virus (LPAIV) H5N9. Wild mallards (n = 30) were separated into three experimental groups; each manipulated through food availability to a different condition level (−20%, −10%, and normal ±5% original body condition), and captive-bred mallards (n = 10) were maintained at normal condition. We found that wild mallards in normal condition were more susceptible to LPAIV infection, shed higher peak viral loads and shed viral RNA more frequently compared to birds in poor condition. Antibody production did not differ according to condition. We found that wild mallards did not differ from captive-bred mallards in viral intensity and duration of infection, but they did exhibit lower antibody titers and greater variation in viral load. Our findings suggest that reduced body condition negatively influences waterfowl host competence to LPAIV infection. This observation is contradictory to the recently proposed condition-dependent hypothesis, according to which birds in reduced condition would be more susceptible to IAV infection. The mechanisms responsible for reducing host competency among birds in poor condition remain unknown. Our research indicates body condition may influence the maintenance and spread of LPAIV by migrating waterfowl.