Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

Kinetics of cytokine expression during primary human immunodeficiency virus type 1 infection.

In the present study, we have determined the kinetics of constitutive expression of a panel of cytokines [interleukin (IL) 2, IL-4, IL-6, IL-10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha)] in sequential peripheral blood mononuclear cell samples from nine individuals with primary human immunodeficiency virus infection. Expression of IL-2 and IL-4 was barely detected in peripheral blood mononuclear cells. However, substantial levels of IL-2 expression were found in mononuclear cells isolated from lymph node. Expression of IL-6 was detected in only three of nine patients, and IL-6 expression was observed when transition from the acute to the chronic phase had already occurred. Expression of IL-10 and TNF-alpha was consistently observed in all patients tested, and levels of both cytokines were either stable or progressively increased over time. Similar to IL-10 and TNF-alpha, IFN-gamma expression was detected in all patients; however, in five of nine patients, IFN-gamma expression peaked very early during primary infection. The early peak in IFN-gamma expression coincided with oligoclonal expansions of CD8+ T cells in five of six patients, and CD8+ T cells mostly accounted for the expression of this cytokine. These results indicate that high levels of expression of proinflammatory cytokines are associated with primary infection and that the cytokine response during this phase of infection is strongly influenced by oligoclonal expansions of CD8+ T cells.

Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.

Ação da proteína amiloide sérica A em melanomas

Concentrações séricas basais da proteína amiloide sérica A (SAA) estão significativamente aumentadas em pacientes com câncer e alguns autores sugerem uma relação causal. Trabalho anterior do grupo mostrou que a SAA induz a proliferação de duas linhagens de glioblastoma humano e afeta os processos de invasividade in vitro, sustentando um papel pró-tumoral para esta proteína. Com base nesse trabalho, investigamos a abrangência dos efeitos de SAA para outro tipo de célula tumoral e para isso escolhemos um painel de linhagens de melanoma humano e uma linhagem primária obtida a partir de aspirado de linfonodo de paciente com melanoma, por nós isolada. Observamos que apesar da célula precursora de melanomas, isto é, melanócito, não produzir SAA, todas as linhagens de melanoma produziram a proteína e expressaram alguns dos seus receptores. Além disso, quando estas células foram estimuladas com SAA houve uma inibição da proliferação em tempos curtos de exposição (48 horas) e efeitos citotóxicos após um tempo maior (7 dias). A SAA também afetou processos de invasividade e a produção das citocinas IL-6, IL-8 e TNF-α. Aos avaliarmos o efeito da SAA na interação das células de melanoma com células do sistema imune, vimos que a SAA ativou uma resposta imune anti-tumoral aumentando a expressão de moléculas co-estumolatórias, como CD69 e HLA-DR, e sua função citotóxica. Ainda, vimos que a produção de TNF-α, IFN-γ, IL-10, IL-1β e IL-8 estimuladas por SAA podem contribuir com os efeitos desta. De forma geral estes resultados nos levam a crer que a SAA tem atividade anti-tumoral em melanomas. Finalizando, com base na importância do desenvolvimento da resistência às terapias atuais para o melanoma, observamos que em células resistentes ao PLX4032, um inibidor de BRAF, os efeitos imunomodulatórios induzidos pela SAA estão abolidos, possivelmente identificando um novo componente da resistência.

Análise da expressão da proteína NY-ESO-1 no melanoma cutâneo

INTRODUÇÃO: o câncer é a doença que mais mata pessoas com idade abaixo de 85 anos e é um problema de saúde pública. Os tumores podem expressar em determinada fase de seu desenvolvimento proteínas anômalas que podem ser alvo de métodos diagnósticos e de intervenções terapêuticas. A expressão de NY-ESO-1 é detectada em 20 a 40% dos melanomas. Há evidências que esta expressão é mais freqüente em tumores de estágios mais avançados e está associada a um pior prognóstico. OBJETIVOS: determinar a frequência de expressão da proteína NY-ESO-1 no melanoma cutâneo e tentar correlacioná-la com o índice de Breslow, aspectos histopatológicos do melanoma, incluindo o infiltrado linfocítico tumoral, e a morbi-mortalidade dos pacientes. MÉTODOS: o presente estudo é longitudinal de coorte retrospectiva e foi realizado de agosto de 2009 a outubro de 2015. Foram selecionados 89 melanomas de 87 pacientes do Ambulatório de Tumores do Departamento de Dermatologia da FMUSP, divididos em 3 grupos, sendo: grupo 1: 34 melanomas com índice de Breslow <= 1,0 mm; grupo 2: 29 melanomas com índice de Breslow entre 1,1 - 4,0 mm e grupo 3: 26 melanomas com índice de Breslow >= 4,0 mm. As lâminas dos exames anátomo-patológicos destes pacientes foram revisadas quanto ao diagnóstico de melanoma, seu índice de Breslow e a presença de infiltrado linfocítico tumoral. A seguir, realizou-se exame de imunohistoquímica para a determinação da presença do antígeno NY-ESO-1 em todos os 89 tumores coletados e em mais 20 nevos (11 displásicos e 9 intradérmicos) escolhidos ao acaso. Através da revisão dos dados do prontuário, foram obtidos os dados clínicos de: idade, sexo, raça, fototipo da pele, local de aparecimento do melanoma, status do linfonodo sentinela quando realizado, desenvolvimento de metástases e sobrevida dos pacientes. Os dados anátomo-patológicos do tumor analisados foram: tipo histológico, presença de ulceração, e tipo de infiltrado linfocítico tumoral. Nos melanomas que apresentavam infiltrado linfocítico tumoral, foram realizados testes imunohistoquímicos para pesquisa de células CD3+, CD8+, FoxP3+ e CD8+FoxP3+ (duplamente positivas). RESULTADOS: O antígeno NY-ESO-1 esteve presente em 19% dos melanomas cutâneos primários e não foi detectado em nenhum dos 20 nevos pesquisados. A expressão do antígeno NY-ESO-1 esteve estatisticamente relacionada a tumores com espessuras maiores. Apresentou também uma associação inversa com o tipo extensivo superficial em relação aos outros tipos histológicos. O infiltrado linfocítico tumoral dos melanomas NY-ESO-1 positivos continha menor número de células CD3+, que se encontravam isoladas ou arranjadas em pequenos grupos de até 5 células, o que contrastava significantemente com os tumores NY-ESO-1 negativos, com maior densidade de células CD3+, dispostas em grandes grupos, com 6 ou mais células. A expressão da proteína NY-ESO-1 não esteve associada à idade, ao sexo, ao fototipo, ao sítio primário do tumor, à presença de ulceração, ao status do linfonodo sentinela, ao desenvolvimento de metástases ou à sobrevida. CONCLUSÕES: Há expressão de NY-ESO-1 em uma porcentagem considerável dos melanomas, principalmente nos mais espessos. O menor número de células CD3+ no infiltrado linfocítico tumoral, acrescido ao fato destas células estarem isoladas ou em pequenos grupos, sugere que embora imunogênico, a expressão do antígeno NY-ESO-1 não resulta num estímulo eficaz do sistema imune no combate ao tumor. O desenvolvimento de uma vacina para estes pacientes poderá, no futuro, aumentar as possibilidades terapêuticas do melanoma

Interleukin-6-deficient mice are highly susceptible to Giardia lamblia infection but exhibit normal intestinal immunoglobulin A responses against the parasite.

In the present study, interleukin-6 (IL-6)-deficient mice were infected with Giardia lamblia clone GS/M-83-H7. Murine IL-6 deficiency did not affect the synthesis of parasite-specific intestinal immunoglobulin A. However, in contrast to wild-type mice, IL-6-deficient animals were not able to control the acute phase of parasite infection. Reverse transcription-PCR-based quantitation of cytokine mRNA levels in peripheral lymph node cells exhibited a short-term up-regulation of IL-4 expression in IL-6-deficient mice that seemed to be associated with failure in controlling the parasite population. This observation suggests a further elucidation of IL-4-dependent, Th2-type regulatory processes regarding their potential to influence the course of G. lamblia infection in the experimental murine host.

Current concepts of tumour metastasis

Background: Tumour metastasis remains the principal cause of treatment failure and poor prognosis in patients with cancer. Recent advances in our understanding of the biology of metastasis are providing novel potential targets for anti-cancer therapies. Aim: This paper reviews the current concepts in tumour metastasis. Methods: A review of Medline publications relating to the molecular biology and therapy of human tumour metastasis was conducted. Results and Discussion: Early metastasis models were based upon the premise of uninterrupted tumour growth, with the inevitable formation of distant metastases and eventual death of the patient. However, current research suggests that metastasis is an inefficient process governed by several rate-limiting steps, and that failure to negotiate these steps can lead to tumour dormancy. Successful metastatic tumour growth depends upon appropriate tumour-host microenvironment interactions and, ultimately, the development of vascularised metastases post-extravasation in the target organ. An understanding of the molecular mechanisms involved in this dynamic process will aid in the identification of therapeutic targets that may allow earlier diagnosis and more specific therapies for patients with metastasis.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen

The mechanism behind the immunostimulatory effect obtained with the cationic liposomal vaccine adjuvant DDA:TDB remains unclear. One of the proposed hypotheses is the 'depot effect' in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. In the present study we devise a method to quantify the in vivo movement of liposomes and vaccine antigen using the radioisotopes H(3) and I(125) respectively. H(3)-labeled liposomes composed of dimethyldioctadecylammonium bromide (DDA) or an 8:1 molar ratio of DDA and trehalose 6,6-dibehenate (TDB) were administered in combination with I(125)-labeled Ag85B-ESAT-6 antigen, both via intramuscular and subcutaneous injection to mice. Furthermore characterisation of the liposomal system in simulated in vivo conditions was undertaken. Our results show that this dual-labeling technique is functional and reproducible. The administration of Ag85B-ESAT-6 without a liposomal carrier leads to rapid dissemination of the antigen from the site of injection. The administration of Ag85B-ESAT-6 together with either DDA or DDA:TDB liposomes however leads to deposition of the antigen at the injection site with detectable levels still being present 14 days post injection. Neither the incorporation of TDB nor the route of injection had any significant influence on the depot effect of DDA-based liposomes. The presence of TDB in DDA liposomes improves draining of liposomes to the lymph node in addition to increasing monocyte influx to the site of injection as highlighted by the intensive blue colouring of the injection site after pontamine blue staining of phagocytic cells in vivo. Our findings provide conclusive evidence for a cationic liposome-mediated deposition of antigen at the injection site with improved monocyte infiltration.

The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production

The use of cationic liposomes as experimental adjuvants for subunit peptide of protein vaccines is well documented. Recently the cationic liposome CAF01, composed of dimethyldioctadecylammonium (DDA) and trehalose dibehenate (TDB), has entered Phase I clinical trials for use in a tuberculosis (TB) vaccine. CAF01 liposomes are a heterogeneous population with a mean vesicle size of 500 nm; a strong retention of antigen at the injection site and a Th1-biassed immune response are noted. The purpose of this study was to investigate whether CAF01 liposomes of significantly different vesicle sizes exhibited altered pharmacokinetics in vivo and cellular uptake with activation in vitro. Furthermore, the immune response against the TB antigen Ag85B-ESAT-6 was followed when various sized CAF01 liposomes were used as vaccine adjuvants. The results showed no differences in vaccine (liposome or antigen) draining from the injection site, however, significant differences in the movement of liposomes to the popliteal lymph node were noted. Liposome uptake by THP-1 vitamin D3 stimulated macrophage-like cells did not show a liposome size-dependent pattern of uptake. Finally, whilst there were no significant differences in the IgG1/2 regardless of the liposome size used as a delivery vehicle for Ag85B-ESAT-6, vesicle size has a size dependent effect on cell proliferation and IL-10 production with larger liposomes (in excess of 2 µm) promoting the highest proliferation and lowest IL-10 responses, yet vesicles of ~500 nm promoting higher IFN-? cytokine production from splenocytes and higher IL-1ß at the site of injection.

Cationic liposomes as adjuvants for subunit protein vaccines:their role in the depot effect and antigen processing by APCs

Introduction: The requirement of adjuvants in subunit protein vaccination is well known yet their mechanisms of action remain elusive. Of the numerous mechanisms suggested, cationic liposomes appear to fulfil at least three: the antigen depot effect, the delivery of antigen to antigen presenting cells (APCs) and finally the danger signal. We have investigated the role of antigen depot effect with the use of dual radiolabelling whereby adjuvant and antigen presence in tissues can be quantified. In our studies a range of cationic liposomes and different antigens were studied to determine the importance of physical properties such as liposome surface charge, antigen association and inherent lipid immunogenicity. More recently we have investigated the role of liposome size with the cationic liposome formulation DDA:TDB, composed of the cationic lipid dimethyldioctadecylammonium (DDA) and the synthetic mycobacterial glycolipid trehalose 6,6’-dibehenate (TDB). Vesicle size is a frequently investigated parameter which is known to result in different routes of endocytosis. It has been postulated that targeting different routes leads to different intracellular signaling pathway activation and it is certainly true that numerous studies have shown vesicle size to have an effect on the resulting immune responses (e.g. Th1 vs. Th2). Aim: To determine the effect of cationic liposome size on the biodistribution of adjuvant and antigen, the ensuing humoral and cell-mediated immune responses and the uptake and activation of antigen by APCs including macrophages and dendritic cells. Methods: DDA:TDB liposomes were made to three different sizes (~ 0.2, 0.5 and 2 µm) followed by the addition of tuberculosis antigen Ag85B-ESAT-6 therefore resulting in surface adsorption. Liposome formulations were injected into Balb/c or C57Bl/6 mice via the intramuscular route. The biodistribution of the liposome formulations was followed using dual radiolabelling. Tissues including muscle from the site of injection and local draining lymph nodes were removed and liposome and antigen presence quantified. Mice were also immunized with the different vaccine formulations and cytokine production (from Ag85B-ESAT-6 restimulated splenocytes) and antibody presence in blood assayed. Furthermore, splenocyte proliferation after restimulating with Ag85B-ESAT-6 was measured. Finally, APCs were compared for their ability to endocytose vaccine formulations and the effect this had on the maturation status of the cell populations was compared. Flow cytometry and fluorescence labelling was used to investigate maturation marker up-regulation and efficacy of phagocytosis. Results: Our results show that for an efficient Ag85B-ESAT-6 antigen depot at the injection site, liposomes composed of DDA and TDB are required. There is no significant change in the presence of liposome or antigen at 6hrs or 24hrs p.i, nor does liposome size have an effect. Approximately 0.05% of the injected liposome dose is detected in the local draining lymph node 24hrs p.i however protein presence is low (<0.005% dose). Preliminary in vitro data shows liposome and antigen endocytosis by macrophages; further studies on this will be presented in addition to the results of the immunisation study.

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.