CD44 and EpCAM: cancer-initiating cell markers.

Embryonic stem cells are immortal, can self renew, and differentiate into all cells of the body. The adult organism maintains adult stem cells in regenerative organs that can differentiate into all cells of the respective organ. Virchow's hypothesis that cancer may arise from embryonic-like cells has received strong support, as it was demonstrated that tumors contain few cells, known as cancer stem or cancer-initiating cells (CIC), that account for primary and metastatic tumor growth. CIC are mostly defined by expression of CIC-markers that are associated and correlated with the potential of CIC to grow in xenogeneic mice. CIC marker profiles have been elaborated for many tumors, with several markers as CD24, CD44, CD133, CD166, EpCAM, and some integrins, being expressed by tumors of different histological type. Their function in promoting CIC maintenance and activity is largely unknown. The fate of stem cells, determined by their position, is minutely regulated by few adjacent cells creating a niche. CIC also require a niche, mostly for settlement and growth in distant organs. This so called pre-metastatic niche is initiated by the primary tumor before metastasizing cell arrival. How do CIC prepare the pre-metastatic niche? Cancer cells secrete a matrix that serves a cross-talk with surrounding tissues. Additionally, cancer cells can abundantly deliver exosomes, which function as long-distance intercellular communicators. Studies on a rat pancreatic adenocarcinoma support our hypothesis that tumor-derived matrix and exosomes are the main actors in forming the pre-metastatic niche with CIC markers being engaged in matrix preparation and/or exosome delivery.

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Activation-induced internalization differs for the tetraspanins CD9 and Tspan8: Impact on tumor cell motility.

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8-CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8-CD9-CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8-INS2-CD49d-clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.

Aldehyde dehydrogenase activity among primary leukemia cells is associated with stem cell features and correlates with adverse clinical outcomes.

OBJECTIVE: Animal models have provided evidence for the existence of leukemia stem cells (LSC). However, prospective isolation of human LSC from patients with acute myeloid leukemia (AML), as well as the assessment of their clinical significance, has remained a major challenge. MATERIALS AND METHODS: We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)), which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS: The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients, the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; p<0.01), a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; p<0.05), higher numbers of long-term culture-initiating cell colonies in vitro (n=25; p<0.01), and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; p<0.05). Above all, we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (p<0.05). CONCLUSION: A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome.