Cell fusion potentiates tumor heterogeneity and reveals circulating hybrid cells that correlate with stage and survival.

High lethality rates associated with metastatic cancer highlight an urgent medical need for improved understanding of biologic mechanisms driving metastatic spread and identification of biomarkers predicting late-stage progression. Numerous neoplastic cell intrinsic and extrinsic mechanisms fuel tumor progression; however, mechanisms driving heterogeneity of neoplastic cells in solid tumors remain obscure. Increased mutational rates of neoplastic cells in stressed environments are implicated but cannot explain all aspects of tumor heterogeneity. We present evidence that fusion of neoplastic cells with leukocytes (for example, macrophages) contributes to tumor heterogeneity, resulting in cells exhibiting increased metastatic behavior. Fusion hybrids (cells harboring hematopoietic and epithelial properties) are readily detectible in cell culture and tumor-bearing mice. Further, hybrids enumerated in peripheral blood of human cancer patients correlate with disease stage and predict overall survival. This unique population of neoplastic cells provides a novel biomarker for tumor staging, as well as a potential therapeutic target for intervention.

Diabetes Device Interoperability for Improved Diabetes Management.

Scientific and technological advancements have led to the increasing availability and use of sophisticated devices for diabetes management, with corresponding improvements in public health. These devices are often capable of sharing data with a few other specific devices but are generally not broadly interoperable; they cannot work together with a wide variety of other devices. As a result of limited interoperability, benefits of modern diabetes devices and potential for development of innovative new diabetes technologies are not being fully realized. Here we discuss diabetes device interoperability in general, then focus on 4 examples that show how diabetes management could benefit from enhanced interoperability: remote monitoring and data sharing, integrating data from multiple devices to better inform diabetes management strategies, device consolidation, and artificial pancreas development.

Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors.

Aneuploidy, a chromosome content other than a multiple of the haploid number, is a common feature of cancer cells. Whole chromosomal aneuploidy accompanying ongoing chromosomal instability in mice resulting from reduced levels of the centromere-linked motor protein CENP-E has been reported to increase the incidence of spleen and lung tumors, but to suppress tumors in three other contexts. Exacerbating chromosome missegregation in CENP-E(+/-) mice by reducing levels of another mitotic checkpoint component, Mad2, is now shown to result in elevated cell death and decreased tumor formation compared with reduction of either protein alone. Furthermore, we determine that the additional contexts in which increased whole-chromosome missegregation resulting from reduced CENP-E suppresses tumor formation have a preexisting, elevated basal level of chromosome missegregation that is exacerbated by reduction of CENP-E. Tumors arising from primary causes that do not generate chromosomal instability, including loss of the INK4a tumor suppressor and microsatellite instability from reduction of the DNA mismatch repair protein MLH1, are unaffected by CENP-E-dependent chromosome missegregation. These findings support a model in which low rates of chromosome missegregation can promote tumorigenesis, whereas missegregation of high numbers of chromosomes leads to cell death and tumor suppression.

Fusion between hematopoietic and epithelial cells in adult human intestine.

Following transplantation of hematopoietic lineage cells, genetic markers unique to the transplanted cells have been detected in non-hematopoietic recipient cells of human liver, vascular endothelium, intestinal epithelium and brain. The underlying mechanisms by which this occurs are unclear. Evidence from mice suggests it is due in part to fusion between cells of hematopoietic and non-hematopoietic origins; however, direct evidence for this in humans is scant. Here, by quantitative and statistical analysis of X- and Y-chromosome numbers in epithelial and non-epithelial intestinal cells from gender-mismatched hematopoietic cell transplant patients, we provide evidence that transplanted cells of the hematopoietic lineage incorporate into human intestinal epithelium through cell fusion. This is the first definitive identification of cell fusion between hematopoietic cells and any epithelial cell type in humans, and provides the basis for further understanding the physiological and potential pathological consequences of cell fusion in humans.

Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension.

It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.

Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells.

Characterization of the intestinal cancer stem cell marker CD166 in the human and mouse gastrointestinal tract.

BACKGROUND & AIMS: CD166 (also called activated leukocyte cell adhesion molecule [ALCAM]) is a marker of colorectal cancer (CRC) stem cells; it is expressed by aggressive tumors. Although the presence of CD166 at the tumor cell surface has been correlated with shortened survival, little is known about its function and expression in normal intestinal epithelia. METHODS: We characterized the expression pattern of CD166 in normal intestinal tissue samples from humans and mice using immunohistochemisty, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction. Human and mouse intestinal tumors were also analyzed. RESULTS: CD166 was expressed on the surface of epithelial cells within the stem cell niche and along the length of the intestine; expression was conserved across species. In the small intestine, CD166 was observed on crypt-based Paneth cells and intervening crypt-based columnar cells (putative stem cells). A subset of CD166-positive, crypt-based columnar cells coexpressed the stem cell markers Lgr5, Musashi-1, or Dcamkl-1. CD166 was located in the cytoplasm and at the surface of cells within human CRC tumors. CD166-positive cells were also detected in benign adenomas in mice; rare cells coexpressed CD166 and CD44 or epithelial-specific antigen. CONCLUSIONS: CD166 is highly expressed within the endogenous intestinal stem cell niche. CD166-positive cells appear at multiple stages of intestinal carcinoma progression, including benign and metastatic tumors. Further studies should investigate the function of CD166 in stem cells and the stem cell niche, which might have implications for normal intestinal homeostasis. CD166 has potential as a therapeutic target for CRC.

Requirements for NuMA in maintenance and establishment of mammalian spindle poles.

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.

Aneuploidy acts both oncogenically and as a tumor suppressor.

An abnormal chromosome number, aneuploidy, is a common characteristic of tumor cells. Boveri proposed nearly 100 years ago that aneuploidy causes tumorigenesis, but this has remained untested due to the difficulty of selectively generating aneuploidy. Cells and mice with reduced levels of the mitosis-specific, centromere-linked motor protein CENP-E are now shown to develop aneuploidy and chromosomal instability in vitro and in vivo. An increased rate of aneuploidy does drive an elevated level of spontaneous lymphomas and lung tumors in aged animals. Remarkably, however, in examples of chemically or genetically induced tumor formation, an increased rate of aneuploidy is a more effective inhibitor than initiator of tumorigenesis. These findings reveal a role of aneuploidy and chromosomal instability in preventing tumorigenesis.

Cell biology: nondisjunction, aneuploidy and tetraploidy.

One simple, widely accepted mechanism for generating an aberrant chromosome number, or aneuploidy, is through nondisjunction--a chromosome distribution error that occurs during mitosis when both copies of a duplicated chromosome are deposited into one daughter cell and none into the other. Shi and King challenge this view, concluding that nondisjunction does not yield aneuploid cells directly, but instead gives rise to tetraploid cells that may subsequently become aneuploid through further division. Here we show that the direct result of chromosome nondisjunction is gain or loss of a single chromosome, which results in near-diploid aneuploidy, not tetraploidy. We suggest that chromatin trapped in the cytokinetic cleavage furrow is the more likely reason for furrow regression and tetraploidization.

Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss.

Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.

Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E.

A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.