von Hippel-Lindau Disease-Associated Hemangioblastomas Are Derived from Embryologic Multipotent Cells

BACKGROUND: To determine the origin of the neoplastic cell in central nervous system (CNS) hemangioblastomas in von Hippel-Lindau disease (VHL) and its role in tumor formation and distribution, we characterized and differentiated neoplastic cells from hemangioblastomas removed from VHL patients. METHODS AND FINDINGS: A total of 31 CNS hemangioblastomas from 25 VHL patients were resected and analyzed. Tumor cells from the hemangioblastomas were characterized, grown, and differentiated into multiple lineages. Resected hemangioblastomas were located in the cerebellum (11 tumors), brainstem (five tumors), and spinal cord (15 tumors). Consistent with an embryologically derived hemangioblast, the neoplastic cells demonstrated coexpression of the mesodermal markers brachyury, Flk-1 (vascular endothelial growth factor-2), and stem cell leukemia (Scl). The neoplastic cells also expressed hematopoietic stem cell antigens and receptors including CD133, CD34, c-kit, Scl, erythropoietin, and erythropoietin receptor. Under specific microenvironments, neoplastic cells (hemangioblasts) were expanded and differentiated into erythrocytic, granulocytic, and endothelial progenitors. Deletion of the wild-type VHL allele in the hematopoietic and endothelial progeny confirmed their neoplastic origin. CONCLUSIONS: The neoplastic cell of origin for CNS hemangioblastomas in VHL patients is the mesoderm-derived, embryologically arrested hemangioblast. The hematopoietic and endothelial differentiation potential of these cells can be reactivated under suitable conditions. These findings may also explain the unique tissue distribution of tumor involvement

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer

BackgroundDisseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs.MethodsWe used fluorescence‐activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA‐Seq) and whole‐exome sequencing after whole genome amplification. We also enriched for BM DTCs with α‐EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT‐PCR) for PCa markers.ResultsAt a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients’ plasma PSA values. RNA‐Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole‐exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA‐Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B‐cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT‐PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%).ConclusionThese data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151343/1/pros23896.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151343/2/pros23896_am.pd

A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers.

Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.We show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators

Oxygen and temperature changes during programmed adjustment of hypoxia.

<p>A single 24-well plate of cell cultures was used and monitored over 48 hrs. (<b>A</b>) The sealed modular hypoxia chamber was placed in cell culture incubator (without a purge with anoxic gas-mix). The chamber was then flushed with the anoxic gas-mix at an initial flow rate of 20 L/min (approx. 2 min) to lower the O₂ level from 21% to 5%. This was followed by a slower flow rate of 5 L/min (approx. 3 min) until O₂% reached 0.5%. (<b>B</b>) expanded view of the change in O₂% during the initial 5 min programmed purge procedure.</p

Required electronic parts^a and accessories^b.

<p>Required electronic parts<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148923#t001fn001" target="_blank">^a</a> and accessories<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148923#t001fn002" target="_blank">^b</a>.</p

Oxygen sensor-hypoxia chamber module.

<p>(<b>A</b>) Close-up views of the Arduino Uno microcontroller board and half-sized breadboard mounted on acrylic plate. <b>(B)</b> Layout of the modular hypoxia chamber (with the oxygen sensor mounted on half-sized breadboard), the Arduino Uno microcontroller and the wire harness routed through one of the gas-flush tube ports, before assembly of the module (lid not shown). 30 AWG silicone-coated (orange colored) wires (cat. no. 2001, adafuit.com) were used in this photograph to clearly illustrate the wire harness.</p

Screen-capture images of CoolTerm terminal window.

<p><b>Upper panel</b>: CoolTerm quick access bar and ribbon; <b>middle panel</b>: format of the initial data output from CoolTerm; <b>lower panel</b>: format of the final data saved by CoolTerm with time-stamps (saved and opened as a Microsoft Notepad file).</p

Outline of the wiring diagram.

<p>A Fritzing (fritzing.org) sketch is presented outlining the wiring connections between the microcontroller, the logic converter, and the oxygen sensor.</p

Representative oxygen and temperature data transmitted by the sensor under experimental conditions.

<p>(<b>A</b>) change in chamber O₂% upon a single 4 min (20 L/min) anoxic gas purge conducted in the presence of cell cultures. (<b>B</b>) lack of a rapid initial increase in O₂% when chamber is devoid of any cell cultures, liquid media or water. (<b>C</b>) change in chamber O₂% with a post-1 day gas purge (in the presence of cell cultures). (<b>D</b>) change in chamber O₂% with daily gas purges in the presence of cell cultures. (<b>E</b>) pressure profile of experiment listed in <b>D</b>. (<b>F</b>) change in chamber O₂% profile upon inclusion of ten cell culture plates.</p