Extracellular lipidosomes containing lipid droplets and mitochondria are released during melanoma cell division.

BACKGROUND: The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues. RESULTS: We report here a new type of large CD133+ EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised. CONCLUSION: Given the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133+ EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.

Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: Potential application in cancer and HIV-1 infection.

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure-activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-G-pseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

Colorectal cancer-derived small extracellular vesicles induce TGFß1-mediated epithelial to mesenchymal transition of hepatocytes.

BACKGROUND: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown. METHODS: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment. RESULTS: Our study shows for the first time that TGFß1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFß1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. CONCLUSIONS: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Anti-human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell-derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-melanoma therapeutic strategies.

VAMP-associated protein-A and oxysterol-binding protein-related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum.

The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes-the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum-localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein-related protein 3 (ORP3), and late endosome-associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated.

Extracellular Vesicles as Biological Shuttles for Targeted Therapies.

The development of effective nanosystems for drug delivery represents a key challenge for the improvement of most current anticancer therapies. Recent progress in the understanding of structure and function of extracellular vesicles (EVs)-specialized membrane-bound nanocarriers for intercellular communication-suggests that they might also serve as optimal delivery systems of therapeutics. In addition to carrying proteins, lipids, DNA and different forms of RNAs, EVs can be engineered to deliver specific bioactive molecules to target cells. Exploitation of their molecular composition and physical properties, together with improvement in bio-techniques to modify their content are critical issues to target them to specific cells/tissues/organs. Here, we will discuss the current developments in the field of animal and plant-derived EVs toward their potential use for delivery of therapeutic agents in different pathological conditions, with a special focus on cancer.

Extracellular Vesicles from Thyroid Carcinoma: The New Frontier of Liquid Biopsy.

The diagnostic approach to thyroid cancer is one of the most challenging issues in oncology of the endocrine system because of its high incidence (3.8% of all new cancer cases in the US) and the difficulty to distinguish benign from malignant non-functional thyroid nodules and establish the cervical lymph node involvement during staging. Routine diagnosis of thyroid nodules usually relies on a fine-needle aspirate biopsy, which is invasive and often inaccurate. Therefore, there is an urgent need to identify novel, accurate, and non-invasive diagnostic procedures. Liquid biopsy, as a non-invasive approach for the detection of diagnostic biomarkers for early tumor diagnosis, prognosis, and disease monitoring, may be of particular benefit in this context. Extracellular vesicles (EVs) are a consistent source of tumor-derived RNA due to their prevalence in circulating bodily fluids, the well-established isolation protocols, and the fact that RNA in phospholipid bilayer-enclosed vesicles is protected from blood-borne RNases. Recent results in other types of cancer, including our recent study on plasma EVs from glioblastoma patients suggest that information derived from analysis of EVs from peripheral blood plasma can be integrated in the routine diagnostic tumor approach. In this review, we will examine the diagnostic and prognostic potential of liquid biopsy to detect tumor-derived nucleic acids in circulating EVs from patients with thyroid carcinoma.

The HDAC6 Inhibitor Tubacin Induces Release of CD133+ Extracellular Vesicles From Cancer Cells.

Tumor-derived extracellular vesicles (EVs) are emerging as an important mode of intercellular communication, capable of transferring biologically active molecules that facilitate the malignant growth and metastatic process. CD133 (Prominin-1), a stem cell marker implicated in tumor initiation, differentiation and resistance to anti-cancer therapy, is reportedly associated with EVs in various types of cancer. However, little is known about the factors that regulate the release of these CD133+ EVs. Here, we report that the HDAC6 inhibitor tubacin promoted the extracellular release of CD133+ EVs from human FEMX-I metastatic melanoma and Caco-2 colorectal carcinoma cells, with a concomitant downregulation of intracellular CD133. This effect was specific for tubacin, as inhibition of HDAC6 deacetylase activity by another selective HDAC6 inhibitor, ACY-1215 or the pan-HDAC inhibitor trichostatin A (TSA), and knockdown of HDAC6 did not enhance the release of CD133+ EVs. The tubacin-induced EV release was associated with changes in cellular lipid composition, loss of clonogenic capacity and decrease in the ability to form multicellular aggregates. These findings indicate a novel potential anti-tumor mechanism for tubacin in CD133-expressing malignancies. J. Cell. Biochem. 118: 4414-4424, 2017. © 2017 Wiley Periodicals, Inc.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells.

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Biochemical and biological characterization of exosomes containing prominin-1/CD133.

Exosomes can be viewed as complex "messages" packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.

Spontaneous formation of tumorigenic hybrids between breast cancer and multipotent stromal cells is a source of tumor heterogeneity.

Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

Prominin-1 (CD133) and Metastatic Melanoma: Current Knowledge and Therapeutic Perspectives.

Innovative approaches to specifically target the melanoma subpopulation responsible for local invasion and metastatic dissemination are needed. Prominin-1 (CD133) expression has been observed in many melanoma cell lines, as well as in primary and metastatic melanomas from patients. Although its function(s) in melanoma is presently unknown, prominin-1 may represent a molecular target, due to its association with melanoma stem cells and with the metastatic phenotype.

Horizontal Transfer of Malignant Traits and the Involvement of Extracellular Vesicles in Metastasis.

Metastases are responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing the metastatic process. Traditionally, cancer metastasis is attributed to the dissemination of neoplastic cells from the primary tumor to distant organs through blood and lymphatic circulation. A thorough understanding of the metastatic process is essential to develop new therapeutic strategies that improve cancer survival. Since Paget's original description of the "Seed and Soil" hypothesis over a hundred years ago, alternative theories and new players have been proposed. In particular, the role of extracellular vesicles (EVs) released by cancer cells and their uptake by neighboring cells or at distinct anatomical sites has been explored. Here, we will outline and discuss these alternative theories and emphasize the horizontal transfer of EV-associated biomolecules as a possibly major event leading to cell transformation and the induction of metastases. We will also highlight the recently discovered intracellular pathway used by EVs to deliver their cargoes into the nucleus of recipient cells, which is a potential target for novel anti-metastatic strategies.

HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells.

The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4+ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7+ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4+ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle.

The intrinsic fusogenicity of glioma cells as a factor of transformation and progression in the tumor microenvironment.

We have previously reported in vitro heterotypic fusion of glioma cells with neural progenitor cells, producing tetraploid cells expressing genetic complements of each partner. Herein, we investigated the fusogenicity of glioma cells. In 1:1 cocultures of single-labeled cells, U87MG cells presented high frequency of homotypic fusogenic events, producing cells that coexpressed both fluorescent proteins. Six percent of the total cells had 8n DNA content, consistent with the finding that the double-labeled cells were actively proliferating. In coculture with fibroblasts, glioma cell fusogenicity resulted in viable reprogrammed cells, thus emerging as a plausible source of tumor cell heterogeneity. As for heterotypic fusion to happen, glioma cells have to establish direct contact with other cells, the effect of stroma on glioma cells was analyzed. Proliferation assays and array analysis of cancer-related pathways established a promalignant effect of stroma. This effect was mediated by fibronectin and was nearly completely abolished by inhibitors of the epidermal-growth-factor receptor. That stroma elicited transduction signaling through the mitogen-activated-protein kinase/extracellular-signal-regulated kinase pathway, which is linked to increased tumor cell migration through extracellular matrix, suggested that glioma cells may actively approach nontumor cells in stromal niches. According to these results, the fusogenicity of glioma cells emerged as an inherent factor for phenotypic changes leading to glioma progression.

Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo.

Radiotherapy represents a first-line treatment for many inoperable lung tumors. New technologies offer novel opportunities for the treatment of lung cancer with the administration of higher doses of radiation in smaller volumes. Because both therapeutic and toxic treatment effects are dose-dependent, it is important to identify a minimal dose protocol for each individual patient that maintains efficacy while decreasing toxicity. Cancer stem cells sustain tumor growth, promote metastatic dissemination, and may give rise to secondary resistance. The identification of effective protocols targeting these cells may improve disease-free survival of treated patients. In this work, we evaluated the existence of individual profiles of sensitivity to radiotherapy in patient-derived cancer stem cells (CSCs) using both in vitro and in vivo models. Both CSCs in vitro and mice implanted with CSCs were treated with radiotherapy at different dose intensities and rates. CSC response to different radiation doses greatly varied among patients. In vitro radiation sensitivity of CSCs corresponded to the therapeutic outcome in the corresponding mouse tumor model. On the other side, the dose administration rate did not affect the response. These findings suggest that in vitro evaluation of CSCs may potentially predict patients' response, thus guiding clinical decision.

Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells.

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand-receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.

Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line.

The phenotypic diversity of breast carcinoma may be explained by the existence of a sub-population of breast cancer cells, endowed with stem cell-like properties and gene expression profiles, able to differentiate along different pathways. A stem cell-like population of CD44(+)CD24(-/low) breast cancer cells was originally identified using cells from metastatic pleural effusions of breast carcinoma patients. We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, human MA-11 breast carcinoma cells acquired increased tumorigenicity and lost CD24 expression compared with the parental cell line. We now report that upon passage of MA-11 mammospheres into serum-supplemented cultures, CD24 expression was restored; the rapid increase in CD24 expression was consistent with up-regulation of the antigen, and not with in vitro selection of CD24(+) cells. In tumors derived from subcutaneous injection of MA-11 mammospheres in athymic nude mice, 76.1+/-9.7% of cells expressed CD24, vs. 0.5+/-1% in MA-11 cells dissociated from mammospheres before injection. The tumorigenicity of sorted CD44(+)CD24(-) and CD44(+)CD24(high) MA-11 cells was equal. Single cell-sorted CD24(-) and CD24(high) MA-11 gave rise in vitro to cell populations with heterogeneous CD24 expression. Also, subcutaneous tumors derived from sorted CD24(-) sub-populations and single-cell clones had levels of CD24 expression similar to the unsorted cells. To investigate whether the high expression of CD24 contributed to the tumorigenic potential of MA-11 cells, we silenced CD24 by shRNA. CD24 silencing (95%) resulted in no difference in tumorigenicity upon s.c. injection in athymic nude mice compared with mock-transduced MA-11 cells. Since CD24 silencing was maintained in vivo, our data suggest that the level of expression of CD24 is associated with but does not contribute to tumorigenicity. We then compared the molecular profile of the mammospheres with the adherent cell fraction. Gene expression profiling revealed that the increased tumorigenicity of MA-11 mammospheres was associated with changes in 10 signal transduction pathways, including MAP kinase, Notch and Wnt, and increased expression of aldehyde dehydrogenase, a cancer-initiating cell-associated marker. Our data demonstrate that (i) the level of CD24 expression is neither a stable feature of mammosphere-forming cells nor confers tumorigenic potential to MA-11 cells; (ii) cancer-initiating cell-enriched MA-11 mammospheres have activated specific signal transduction pathways, potential targets for anti-breast cancer therapy.