Cancer is the 2nd most common cause of death among children after accidents.
Childhood cancer is an umbrella term for many other types of this disease. Every September, many charities, researchers and parents of children with cancer work hard to raise awareness of this cancer. You may learn more about kids with cancer, their loving families, the doctors and caregivers who look after them and treat them, the young survivors of cancer and those kids and teens who lost their battle, and the scientists who work hard to find a way to stop childhood cancer.
This year, our research team will run the Pub Quiz on September 18th, 2024, in honour of Childhood Cancer Awareness Month. All donations will go to the Conor Foley Neuroblastoma Research Foundation (CFNRF).
If you would like to get involved in this amazing challenge and help us raise vital funds for childhood cancers, you can contribute to our fundraising page:
HDAC inhibitors are drugs that target specific enzymes involved in gene regulation. This study tested broad-spectrum HDAC inhibitors as standalone treatments and combined them with doxorubicin, a well-known chemotherapy drug.
But why on Zebrafish? The zebrafish model provides a rapid and efficient means of testing these treatments, offering valuable insights into their potential use in combating neuroblastoma. This model allows for assessing drug efficacy and helps understand the associated toxicities quickly, making it a powerful tool for developing new anti-cancer therapies.
In the study, fish larvae were implanted with fluorescently labelled, well-established neuroblastoma cell line (SK-N-BE(2)-C) and patient samples (HD-N33, NB-S-124) to grow tumours. Non-cancerous cells (VH7 fibroblasts) were utilized to verify that tumour progression in zebrafish was specific to tumour cells. The engraftment of human cells into fish larvae was confirmed by immunohistochemistry (IHC) staining on zebrafish sections injected with neuroblastoma cells (SK-N-BE). This was achieved using a STEM121 antibody that reacts specifically with a human cytoplasmic protein. The findings showed that pediatric tumour cells survive and grow in the zebrafish model at rates like those observed in human tumours.
Before testing drug efficacy in zebrafish xenografts, optimal drug concentrations and maximal tolerated doses (MTD) were determined. Toxicity tests were conducted by treating fish larvae cells for three days without tumour cell injection to identify the maximum tolerated dose that did not cause observable morbidity, changes in morphology, or severe aberrations in larval behaviour. and lethal dose (LD) for each compound. To find optimal drug concentrations, larvae with xenografted tumour cells were incubated with increasing drug doses 24 hours post-implantation to the maximally tolerated dose (MTD). The relative IC50 values were then determined based on changes in tumour mass volume.
To evaluate the treatment, SK-N-BE(2)- cells were used to test the broad-spectrum HDAC inhibitors, including panobinostat, vorinostat, and tubastatin A, both alone and combined with doxorubicin. The partial response rate (PR) was measured to see how well different drug combinations work to shrink tumours in the zebrafish model. Here’s what they found: Doxorubicin combined with panobinostat resulted in a 23% PR, Doxorubicin combined with tubastatin A showed a 31% PR, and Doxorubicin combined with vorinostat achieved the best result with a 36% PR.
To test the effectiveness of the HDAC inhibitor treatment, they monitored the tumour growth using a confocal microscope before and 48 hours after giving the drug. The test revealed that a 48-hour treatment of SK-N-BE (2)-C zebrafish xenografts with vorinostat and doxorubicin alone, `and in combination, increased cell death. The combination of these two drugs was the most effective, causing a significant increase in cancer cell death (apoptosis) by decreasing cell proliferation, as indicated by reduced PPH3 marker and activating the number of Cleaved caspase-3 (Figure 1).
Figure 1: Treatment for 48 h with Vorinostat, doxorubicin, or a combination of both increased the amount of cleaved caspase-3 and reduced mitotic tumour cells. Adapted from Pharmaceuticals2020, 13(11), 345
In essence, this study validates the use of HDAC inhibitors in treating neuroblastoma and paves the way for broader applications of zebrafish models in cancer research. As we look to the future, these innovative models could significantly enhance our ability to develop effective cancer therapies, making strides towards better treatments and, ultimately, more effective cures.
This article by Krieger et al. discusses the most common form of brain cancer called glioblastoma. Due to its highly aggressive nature, research must be conducted consistently and rapidly to develop new treatments. This has proven challenging due to primary tumours being resected before further research can be done, as well as the lack of current technologies to fully explore relationships between GBM and surrounding brain tissues. This study aimed to study the aforementioned interactions in under 4 weeks, accounting for the rapid progression of the disease in real life.
GBM cells were first derived from four patients and treated with glutamine, heparin, epidermal and fibroblast growth factors, then underwent a sequence of manipulations, such as second-generation replication lentivirus infection of GBM cells, iPSC line 409b2 inoculation in Aggrewell plates and later manipulation with invasion assays, and scRNA sequencing, which, along with the Aggrewell cells, produced neural progenitor cell spheroids for analysis. Confocal microscopy and the developed image processing algorithm allowed for visualization of these cells following fluoroscopy and depicted consistent growth of tumour cells. There was also the growth of microtubules. Any dissociated organoids were then co-cultured with GBM cells again, promoting interaction between the two. Further analysis revealed the upregulation of 45 genes, including PAX6, GJA1, GPC3, and others involved in cell regulation.
In conclusion, this novel mechanism of analysis of GBM cells using Aggrewell plates provided fruitful results, indicating intricate relationships between GBM cells and organoids, providing crucial insight for treatments by elucidating specific gene expression, heterogeneity of cells, and offering new targets based on ligand-receptor interactions. The particular relevance of this study to my work is regarding the usage of Aggrewell plates, which I am currently studying to determine how best to keep cells growing successfully within the wells. This article proves the usability and efficiency of Aggrewell and establishes its crucial role in the realm of brain cancer treatment research.
How do researchers study cells? How do we get the nitty gritty?
We use many methods to tag and chase various cell components. One of my favourites is fluorescent microscopy. It allows the use of nearly all spectrum of colours from blue to purple in one go. However, we prefer to narrow it down to 2-3 colours and avoid their overlap.
How does it work? First, we use DAPI or Hoescht, which are blue fluorescent dyes used to stain DNA. This way, we tag the nucleus of the cell. Then, we tag a protein of interest. In our case, it was MYCN, a protein that acts as a transcription factor. MYCN amplification is associated with poor prognosis in neuroblastoma. As a transcription factor, it binds to genomic DNA and is located in the nucleus. We used a specific antibody that was labelled with a green fluorescent dye. Look at the image below. The green colour pattern overlaps with the blue colour. Then, we tagged the cytoskeleton, a complex of various proteins that hold the cell architecture and dynamics. We used phalloidin with red fluorescence. It is a highly selective bicyclic peptide and a popular choice for staining actin filaments.
Neuroblastoma organoids stained with DAPI, Phalloidin and anti-MYCN antibody. This work was done during the Fulbright journey to Ewald’s Lab at Johns Hopkins
Now, we can enjoy visualising cells and test different research questions. For example, how do cells respond to a drug? Or how do neuroblastoma cells spread?
Huge congrats to a newly minted Dr Ciara Gallagher! She defended her PhD on March 8 – International Women’s Day. Your enthusiasm and perseverance are truly fascinating! May this be the stepping stone towards a brighter future, Ciara!
We thank examiners Dr Marie McIlroy (RCSI) and Prof Jan Škoda (Masaryk Uni) for the time and expertise they provided.
We also thank the Irish Research Council for their generous support!
Dr Ciara Murphy (Chair), Dr Olga Piskareva (Supervisor), Dr Ciara Gallagher, Prof Jan Skoda (examiner), Dr Marie McIlroy (Examiner)
Researchers use various methods, but I employ gene knockdown in my experiments. Basically, I use small RNA molecules that specifically target and degrade the mRNA of my gene of interest. This leads to a decrease in the corresponding protein levels, enabling me to observe the effects on neuroblastoma cell behaviour.
I feel a bit like Sherlock Holmes, you know? I’m selectively putting my suspect protein – the one I’m eyeing – under the spotlight to see how it’s pulling the strings on the cell’s behaviour. It’s like I’m in a cellular mystery, complete with a gene knockout magnifying glass 🔍🧬🕵
So, what I’ve been up to these past months is knocking down my protein and trying to find answers to the following questions:
Can neuroblastoma cells survive? And if not, how do they meet their demise? Do they go on a growth spree and start proliferating? Are they capable of migration? And here’s the twist – when my protein of interest takes a dip, do other proteins decide to change their expression levels?
The picture below can probably help you get an idea of what I’ve done so far. Do you see those brighter spots in Pictures A and B? Those are dead cells. Their number indicates the proportion of dead cells after a treatment. Picture A has just a few; the majority are healthy and well-spread cells. This is our negative control, a condition when we show neuroblastoma cells that have been transfected, but no gene knockdown happened. Transfection is the term for introducing small RNA molecules. Now, in Picture B, when we knocked down the protein, it caused the death of the cells, and you can clearly see that from all those many little bright spots.
We have found answers to many of the previous questions, but new questions have arisen, and we can’t wait to answer them!
A wonderful day of knitting – Knit-A-Thon-2023 raised 913 euros. A massive thank you to everyone who stopped by and donated on the day and beyond. Every cent counts! The money was split evenly between our four chosen charities: The Conor Foley Neuroblastoma Research Foundation (CFNRF), Neuroblastoma UK (NBUK), Oscars Kids and Childhood Cancer Ireland (CCI). These charities were established and are run by parents, some of whom lost their children to cancer. They continue their children’s legacy, doing an amazing job of advocating for children with cancer and better funding for research and aftercare.
Knit-A-Thon 2023
And a special thank you to Ciara’s mam Aggie for the amazing handmade raffle prizes (chromosomes, antibodies, cup holders and many more) and a Master class on the day! We thank Jenny Duffy (RCSI Events and Communications Coordinator) for her time crocheting with us and for us! Thanks to Anggie’s and Jenny’s skills, there were lots of mascots to win – and many of them collected already. We much appreciate the support from the RCSI Estates and Porters who looked after us on the day.
We are the Cancer Bioengineering Group, and September is a very special month for us as it is Childhood Cancer Awareness Month. Childhood cancer is the 2nd leading cause of death in children after accidents. Our group researches childhood cancer neuroblastoma, a cancer of immature nerve cells. Despite intensive multimodal treatment, as many as 1 in 5 children with aggressive neuroblastoma do not respond, and up to 50% of children that do respond experience disease recurrence with many metastatic tumours resistant to many drugs and more aggressive tumour behaviour that all too frequently results in death.
This is what we want to change! We believe that every child deserves a future, and our team of postgraduate researchers led by Dr Olga Piskareva is dedicated to strengthening our knowledge of this disease and identifying new potential ways to tackle it, as well as taking part in fundraising activities so our group and others can continue with this research.
On Tuesday, the 19th of September, we are running a Knit-A-Thon using gold and purple yarn to mark childhood cancer and neuroblastoma, respectively. Our patterns are inspired by Neuroblastoma UK and Mr Google, indeed.
This year, we honour 4 charities that are doing an amazing job of advocating for children with cancer and better funding for research and aftercare. Therefore, the donations we receive will be split equally among The Conor Foley Neuroblastoma Research Foundation (CFNRF), Neuroblastoma UK (NBUK), Oscars Kids and Childhood Cancer Ireland (CCI). If you would like to get involved in the Knit-A-Thon and help us raise vital funds for childhood cancers, come along on the day and make a donation to these wonderful charities.
On the day, RCSI 123 SSG will #GoGold in support of this cause. Please come by to see the RCSI building lit up and share your pictures on social media with the hashtag #ChildhoodCancerAwarenessMonth to raise awareness.
Ready, Steady, Go!
Every year we manage to raise an amazing 1500-2000 euros by organising a new challenge. We are eager to surpass that target this year. All donations no matter how small are appreciated at GoFundMe.
Hi there, Ciara here again, a final-year PhD student in our research group. I can’t believe September has rolled around again, meaning one thing: it’s Childhood Cancer Awareness Month (CCAM). In honour of this month, I would like to tell you a little bit about the childhood cancer we study in our lab and the research that I do to one day help save children from this disease.
Neuroblastoma is an aggressive childhood cancer, with sadly only 20% of late-stage patients surviving after 5 years. Progressive disease and cancer relapse are common in neuroblastoma. This is due to standard treatment regimens not being adequate for treating high-risk patients. Current treatment also may cause a series of adverse reactions in patients. Therefore, my research focuses on developing a 3D model of high-risk neuroblastoma that models the cancer more accurately in a laboratory setting. This will act as a beneficial platform to test whether new therapies effectively fight the patients’ cancer cells, leading to better treatment options for children with neuroblastoma.
Below is a picture of how we grow these cancerous cells on our 3D model and visualise them with fluorescent stains. When we can see them like this under a microscope, we can study how they move and grow to help us understand how to treat them.
Here, we can see the cells growing on our 3D cancer model. This image is magnified by 200 times to be able to see the individual cancer cells. The green stain is the outside of our cancer cells, or we use the term, the cell membrane. The blue is the inside, or as some of you may know the term, the nucleus of the cell. (It is amazing what we can see with the power of microscopes, right?)
As you may know, every year, we support amazing charities by raising vital funds to keep the fight against childhood cancer going. Keep your eyes peeled on our Twitter for updates on what crazy activity we have committed to this year!!
