Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

Background

While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.

Methods

We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.

Findings

Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.

Conclusions and Hypothesis

Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.     

The Relationship between Processing Speed and Regional White Matter Volume in Healthy Young People

Processing speed is considered a key cognitive resource and it has a crucial role in all types of cognitive performance. Some researchers have hypothesised the importance of white matter integrity in the brain for processing speed; however, the relationship at the whole-brain level between white matter volume (WMV) and processing speed relevant to the modality or problem used in the task has never been clearly evaluated in healthy people. In this study, we used various tests of processing speed and Voxel-Based Morphometry (VBM) analyses, it is involves a voxel-wise comparison of the local volume of gray and white, to assess the relationship between processing speed and regional WMV (rWMV). We examined the association between processing speed and WMV in 887 healthy young adults (504 men and 383 women; mean age, 20.7 years, SD, 1.85). We performed three different multiple regression analyses: we evaluated rWMV associated with individual differences in the simple processing speed task, word–colour and colour–word tasks (processing speed tasks with words) and the simple arithmetic task, after adjusting for age and sex. The results showed a positive relationship at the whole-brain level between rWMV and processing speed performance. In contrast, the processing speed performance did not correlate with rWMV in any of the regions examined. Our results support the idea that WMV is associated globally with processing speed performance regardless of the type of processing speed task.     

Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter

Ecdysteroids, primarily 20-hydroxyecdysone (20E) and ecdysone (E), are steroid hormones that regulate various developmental and physiological processes in insects. Commonly, immunoassays are used to quantify ecdysteroid titers of insects. However, the antibodies used in these assays react not only with 20E and E but often also with their inactive reserves and metabolites, and thus require purification before they can be quantified precisely. Here, we developed a simple cell-based method to quantify only the hormonally active ecdysteroids using newly established cells harboring the firefly luciferase gene under the control of the ecdysteroid-inducible promoter of the E75A gene of the silkworm Bombyx mori L. These cells also constitutively expressed the Renilla luciferase gene using the baculovirus ie2 promoter for internal reference. This cell-based method detected hormonally active ecdysteroids with significantly higher sensitivity than their inactive metabolites. Hemolymph ecdysteroid titers, determined using a dual luciferase assay after exposing these cells to crude extracts of B. mori larval and pupal hemolymph, agreed well with the sum of the 20E and E titers, which were quantified individually using a radioimmunoassay after they had been separated by HPLC. Thus, this method is very useful for quantifying the ecdysteroid titers of insects, particularly when the samples contain large amounts of ecdysteroid reserves and metabolites.     

Rapid evaluation of a protein-based voltage probe using a field-induced membrane potential change

The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1–S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.     

Enhanced angiogenic potency of monocytic endothelial progenitor cells in patients with systemic sclerosis

Introduction  

Microvasculopathy is one of the characteristic features in patients with systemic sclerosis (SSc), but underlying mechanisms still remain uncertain. In this study, we evaluated the potential involvement of monocytic endothelial progenitor cells (EPCs) in pathogenic processes of SSc vasculopathy, by determining their number and contribution to blood vessel formation through angiogenesis and vasculogenesis.