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Melbourne, Australia

Cancer cells that lie 'snoozing' in the skeleton can be awakened - or left to slumber on - by changes in the bone that surrounds them, Australian scientists have shown. In a world first, researchers from the Garvan Institute of Medical Research have used state-of-the-art microscopy techniques to watch cancer cells sleep within living bone over a period of months. They show that cancer cells can be 'woken up' when bone tissue is broken down around them, suggesting new possibilities for treating metastatic cancer in bone. In several cancers (including breast and prostate), cancer cells can spread from the original tumour site into bone. Once there, they settle in 'for the long haul', remaining inactive for months or even many years. Eventually, though, some of these cells can 'wake up' and begin dividing, forming secondary cancers (or metastases) in bone and dramatically worsening the prognosis of cancer patients. "Once a cancer spreads to bone, it becomes notoriously difficult to treat," says Professor Peter Croucher, Head of Garvan's Bone Biology Division and the study's lead investigator. "So, it's important to establish exactly what wakes those cells in bone. Is it some signal within the cells themselves, or is it a change in their environment? "You could compare it to how we ourselves wake in the morning. Some of us wake naturally, but others need an external signal, whether it's an alarm clock or sunshine streaming through the window." The research team set out to discover which scenario holds true for cancer cells in bone. Their research is published today in the leading journal Nature Communications. Using a groundbreaking technique called intravital two-photon microscopy, the researchers tracked the fate of sleeping cancer cells in the tibia (the main bone in the long part of the leg) of a living mouse. They introduced cells from multiple myeloma (a cancer of blood cells that arises in bone) into the mouse, and watched as a small number of the cells lodged in the tibia and 'went to sleep'. These vanishingly rare sleeping cells could be detected because they contained a fluorescent dye that was lost rapidly from dividing (wakeful) cells. Dr Tri Phan, who co-led the study, says, "Because we were looking at a long bone like a tibia (rather than the skull, which is more commonly studied), we could watch the same sleeping cancer cells, in the same bone, in the same mouse, over a long period of time - and this is something that hasn't been done before." Prof Croucher says that studying the same set of cells over a period of months gave vital clues about what caused them to reactivate. "Because we've done it this way, we can show that there are a great many dormant cells - yet only some of them get woken up, and those that do wake, wake at different times. We even saw some cells that woke then went back to sleep again. "The fact that these myeloma cells behave so differently, despite coming from the same cancer cell line, gave us our first clue that it is a signal from outside the cells that is controlling when they wake." The next challenge was to work out the precise nature of the 'wake-up call' from bone. Dr Michelle McDonald, a bone biologist on the research team, says, "In this study, we've shown that bone's dynamic process of building up and breaking down can send signals to cancer cells to stay sleeping or to wake. "Cells known as 'osteoblasts' are known to help build new bone, while 'osteoclast' cells break down bone. "We were able to show that myeloma cells are usually kept asleep by close association with a layer of osteoblast-like cells, called bone-lining cells, in the endosteum (an internal surface within bone). The bone-lining cells are essentially inactive, so we can think of them as providing a quiet environment in which myeloma cells sleep undisturbed. "Crucially, we can wake those myeloma cells by activating osteoclasts, which break down bone tissue. We think the osteoclasts are physically changing the local environment of the cancer cells and waking them up in the process - as if they were literally throwing them out of bed. "We know that bone remodelling is going on in all of us - so a myeloma cell could be woken in an essentially random fashion, by having its local environment remodelled by osteoclasts. Essentially, a cancer cell could be woken by being in the wrong place at the wrong time." What does this mean for treating secondary cancers in bone? Prof Croucher says, "Now we can see that the cancer cells are woken by changes in the surrounding bone, we can think in a whole new way about treating bone metastasis - and there are two treatment approaches in particular that have promise. "The first is that we could inhibit the breakdown of bone by osteoclasts so as to keep cancer cells in long-term hibernation. In fact, there are already drugs that can do this, such as bisphosphonates (used to protect bone in individuals with osteoporosis), and there's already evidence that these drugs do improve survival in breast cancer patients. "The other, more radical, option is to do the opposite - to wake the sleeping cells by activating osteoclasts and driving the breakdown of bone. Most cancer treatments target active, dividing cells, so waking the sleeping cells should make them susceptible to those therapies - and, ultimately, could eradicate any residual disease."


Ballantyne K.N.,Office of the Chief Forensic Scientist | Ballantyne K.N.,La Trobe University | Salemi R.,Biology Division | Guarino F.,Biology Division | And 4 more authors.
Australian Journal of Forensic Sciences | Year: 2015

Recent environmental monitoring studies have highlighted a need to confirm cleaning procedures are performing to suitable levels for highly sensitive STR kits such as PowerPlex 21. To ensure that DNA contamination minimisation procedures are adequate, we have investigated the efficacy of sodium hypochlorite and a commercial, non-corrosive alternative, Virkon, at a range of concentrations for their DNA decontamination ability. Cleaning solutions were trialled across a range of body fluids and surface types, to cover the variety of potential contamination circumstances encountered within typical forensic laboratories. Given all factors tested, it was concluded that a 1% solution of sodium hypochlorite, sprayed on the surface and left for 5 min before drying and wiping with 70% ethanol, was able to remove DNA, saliva, blood, semen and skin cells from both smooth and pitted surfaces. However, safety testing revealed that the combination of hypochlorite and ethanol produced levels of gaseous chlorine at or above the recommended exposure limits. Subsequently, a cleaning protocol of 1% hypochlorite followed by distilled water was tested for efficacy, and subsequently introduced throughout the laboratory. © 2015 Australian Academy of Forensic Sciences.


Verdon T.J.,Biology Division | Verdon T.J.,La Trobe University | Mitchell R.J.,La Trobe University | van Oorschot R.A.H.,Biology Division
Forensic Science International: Genetics Supplement Series | Year: 2011

The efficiency of extracting DNA directly from substrates varies according to a range of factors including the type of substrate and the extraction technique. Two routinely used DNA extraction methodologies (automated DNA IQ and manual Chelex) were texts on their efficiency to extract DNA from a range of blood volumes (0.1-30. μL) on plastic and cotton. The efficiency of extracting DNA from plastic appeared to be lower than from cotton for both methods, but was only statistically significant for Chelex extractions. Pairwise comparisons of blood volumes extracted using DNA IQ showed statistically significant differences. Comparisons of Chelex extractions of different blood volumes also showed significant differences for cotton, but not for plastic. The threshold effect of DNA IQ was only demonstrated at blood volumes above 15. μL. This preliminary research highlights discrepancies between extraction methods and demonstrates that laboratories should be aware of the limitations of their analysis techniques, as knowledge of extraction efficiencies may assist in optimisation of methodologies and procedures. Extraction efficiency analysis will also allow for more accurate assessment of the influence of used methodologies in studies relating to determination of DNA transfer rates, and, should these transfer studies be put into practice, in casework. © 2011 Elsevier Ireland Ltd.


Ballantyne K.N.,Biology Division | Ballantyne K.N.,La Trobe University | Van Oorschot R.A.H.,Biology Division | Mitchell R.J.,La Trobe University
Forensic Science International: Genetics | Year: 2011

Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples. © 2010 Elsevier Ireland Ltd. All rights reserved.


Verdon T.J.,Biology Division | Verdon T.J.,La Trobe University | Ballantyne K.N.,Biology Division | Mitchell R.J.,La Trobe University | van Oorschot R.A.H.,Biology Division
Forensic Science International: Genetics Supplement Series | Year: 2011

The Pinpoint DNA Isolation System (Zymo Research) uses a dissolved polymer compound to remove and extract DNA from slide mounted pathology specimens. This polymer is applied to a non-porous substrate on which biological material is deposited, allowed to dry, and the polymer, containing cells and DNA, is peeled off. The polymer dissolves into solution during extraction, theoretically releasing more DNA into the extract than standard cotton swabs; a notion supported by initial data. We performed preliminary experiments to test its effectiveness in comparison to wet/dry swabbing methodology for forensic samples, including replicates each of 10. μL of saliva on glass slides and a pitted, non-porous surface. Results from the glass substrates showed that the initial application method of spreading with pipette tips generated significantly less DNA than the swabbing method. The mode of polymer application is being investigated further with the aim of improving DNA collection. Sampling from a pitted surface with Pinpoint exhibited significantly less variability than swabbing, but the mean quantity of DNA obtained from both collection methods was comparable. The Pinpoint system, combined with an optimised application method, may be another effective way to sample DNA in forensic casework. It has the potential to collect higher quantities of DNA than traditional methods which may be especially advantageous in casework involving trace DNA. © 2011 Elsevier Ireland Ltd.

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